System and Method for Diagnosis and Treatment

ABSTRACT

This invention relates to a low cost rapid response diagnostic system to determine cortisol levels in patients selected as potential candidates for GCR (glucocorticoid receptor) antagonist therapy utilizing a GCR antagonist, such as ORG 34517. The rapid, sensitive, and inexpensive test can be used to determine patients who have non-normal cortisol production or disordered circadian rhythms as a method for selecting subjects for GCR antagonist therapy for whom it is likely to have beneficial and/or therapeutic effects, and can also be used to monitor changes in cortisol levels in response to treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. Ser. No. 15/478,368filed on Apr. 4, 2017, which is a Divisional application of U.S. Ser.No. 15/095,248, filed Apr. 11, 2016, which is a Divisional applicationof U.S. Ser. No. 14/802,060 filed Jul. 17, 2015, now U.S. Pat. No.9,314,473, issued Apr. 19, 2016, which is a Continuation-in-Part of U.S.Ser. No. 14/100,714 filed Dec. 9, 2013, now U.S. Pat. No. 9,114,147,issued Aug. 25, 2015, which is a Divisional application of U.S. Ser. No.13/364,651, filed Feb. 2, 2012, now U.S. Pat. No. 8,658,128, issued Feb.25, 2014, which claims benefit under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application 61/462,492 filed Feb. 3, 2011; U.S.Provisional Patent Application 61/463,212 filed Feb. 14, 2011; U.S.Provisional Patent Application 61/465,703 filed Mar. 23, 2011; U.S.Provisional Patent Application 61/518,248 filed May 3, 2011; and U.S.Provisional Patent Application 61/519,323 filed May 20, 2011, thedisclosures of which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION 1. Field of Invention

This invention relates to a low cost rapid response diagnostic system todetermine cortisol levels in patients selected as potential candidatesfor GCR (glucocorticoid receptor) antagonist therapy utilizing a GCRantagonist, such as ORG 34517. The rapid, sensitive, and inexpensivetest can be used to determine patients who have non-normal cortisolproduction or disordered circadian rhythms as a method for selectingsubjects for GCR antagonist therapy for whom it is likely to havebeneficial and/or therapeutic effects, and can also be used to monitorchanges in cortisol levels in response to treatment.

2. Description of Related Art

ORG 34517 is one of a class of therapeutic agents designed to block theglucorticoid receptor (GR), acting as an antagonist for endogenouscortisol. Its primary developmental pathway has been as a treatment forneuropsychiatric diseases that are characterized by dysregulatedsignaling in the hypothalamic-pituitary-adrenal axis, often with higherthan normal circulating levels of endogenous cortisol. Of particularnote are the phase 2 clinical trials that have been completed for thetreatment of psychotic depression. Other possible uses in this diseasecategory which are under investigation include: post-traumatic stressdisorder, weight gain in patients requiring long term anti-psychoticmedication, hospital delirium of the elderly, etc. In addition, thediverse data indicate a possible role for GR-blockade as a means ofpromoting chemo-sensitization of target tumors. Pre-clinical trialsdemonstrate significant outcomes—breast cancer growth slowed andreversed. These are pre-clinical trials in which the company hassuccessfully demonstrated the efficacy of a chemotherapy sensitizer for“triple negative” breast cancer.

The “triple negative” breast cancer is the most difficult to treat typeof breast cancer, and is indicated by the patient testing negative forestrogen-receptor, progesterone-receptor and her-2/neu. The triplenegative breast cancer is resistant to chemotherapy. Primary drugresistance and early onset of resistance are seen in other tumor types,as well, for example in liver and ovarian cancers, where there is asignificant unmet medical need for effective therapy. Chemotherapy isstill a key approach to cancer treatment. Chemosensitizers wouldcontribute to improve the efficacy of current therapeutic drugs andpotentially improve their side effect profile. The world cancer marketwas estimated at $23 billion in 2004 and is expected to grow to at least$61 billion by 2013 with a CAGR of 14.7%. U.S. Patent ApplicationPublication no. 2011/0269728 (Pan et al.), incorporated by referenceherein in its entirety, discloses a method of killing breast cancercells comprising administering to a breast cancer patient an effectiveamount of a combination of anti-cancer compounds, wherein the anticancercompounds comprise a glucocorticoid receptor antagonist and achemotherapeutic.

The present invention provides a low cost rapid response diagnosticsystem to determine salivary cortisol levels in patients selected aspotential candidates for GCR (glucocorticoid receptor) antagonisttherapy utilizing a GCR antagonist such as ORG 34517. The inventors havedeveloped a saliva based diagnostic device for cortisol detection toaccompany the development of ORG34517 as a therapeutic agent formultiple indications.

Clinical testing of cortisol levels in patients is a high cost,laborious test that can be salivary or serum, with samples taken from apatient and sent to a lab to await results. The cost and time factor forsuch tests has, to date, been prohibitive, preventing the rapidquantitative determination necessary to assign treatment with aglucocorticoid receptor (GCR) antagonist due to the inability to makethe determinations of cortisol levels at point of need or to monitorchanges in cortisol as a measure of treatment response. By allowing thephysician to determine the elevated cortisol level of a patient and inturn provide a therapeutic for such elevation at point of measurement,the physician can qualify the best candidates suited for this type oftherapeutic. The system also enables continual monitoring of the patientduring treatment for assessment of responsiveness to treatment.

The present invention provides a system in which an apparatus uses ahigh void volume carrier to absorb sufficient amounts of saliva to thenbe placed into a reaction vessel with a reagent. The reagent is mixedwith the sample and then is combined with, for example, a fluorescentligand or pigment-labeled ligand and placed into a device to determinesalivary cortisol levels of the patient in less than 5 minutes, ineither a portable, miniaturized fluorescence polarization reader (in theformer case) or into a lateral flow device (in the latter) for measuringamounts of substrate in a small amount of fluid by direct or indirectmethods.

The reader apparatus, for example, provides temperature control andon-board mixing as an aid in viscosity control of the reaction to ensurebetter accuracy and precision.

The invention and method for non-invasive sampling and detecting thepresence of a biological substance of interest in a test sample of, forexample, saliva, or a bodily fluid, combining said test sample with abuffering system (Reagent 1) containing viscosity controllers andstabilizers in a reaction vessel, mix solution well, combining said testsample and buffering system mixture with a fluorescence-labeled ligand(Reagent 2) to said biological substance (assay solution) in a reactionvessel, mix solution well, and detecting a change of the assay solutionin the fluorescence polarization reader, or a pigment labeled ligand.

The present invention relates to the use of glucocorticoid receptor(GCR) antagonists (e.g. ORG 34517) enabled by a device for rapidly,sensitively, specifically quantifying salivary cortisol levels as asurrogate for serum cortisol levels in a low cost manner. One purpose ofthis combination of inventions is to determine patients who havenon-normal cortisol produced by the adrenal cortex or disorderedcircadian rhythms as a method for selecting subjects for GCR antagonisttherapy for whom it is likely to have beneficial and/or therapeuticeffects, i.e., those with abnormal high levels (but maintained circadianrhythm), over responsiveness to normal levels, high night-time cortisollevels as a feature of disrupted circadian rhythm. The rapid, sensitive,and inexpensive test can also be used to monitor changes in cortisollevels in response to treatment, in patients who have non-normalcortisol produced by the adrenal cortex or disordered circadian rhythmsas a method for selecting subjects for GCR antagonist therapy for whomit is likely to have beneficial and/or therapeutic effects, but also inpatients having normal baseline cortisol at the start of treatment, butfor whom changing cortisol levels during treatment will indicateresponsiveness to the GCR antagonist.

The endogenous glucocorticoids are steroids predominantly produced inthe adrenal cortex. Glucocorticoids are important steroids forintermediary metabolism, immune, musculoskeletal, connective tissue andbrain function. The main glucocorticoid in the body is cortisol. Theproduction and secretion of cortisol is governed by a complex and highlyefficient system that includes the hypothalamus, pituitary and theadrenal glands i.e., hypothalamic-pituitary-adrenal axis (HPA). Cortisolsecretion has a circadian release rhythm with peak values in earlymorning and trough values at midnight.

The production and secretion of the most important glucocorticoid,cortisol, is governed by a complex and highly efficient system thatincludes the hypothalamus, pituitary and the adrenal glands i.e.,hypothalamic-pituitary-adrenal axis. Cortisol secretion is regulated bythe suprachiasmatic nucleus of the hypothalamus into a circadian releaserhythm. The timing is synchronized with the solar day by dark-lightshifts, which normally reflect the habitual sleep-wake pattern.Therefore in healthy persons, the cortisol secretion has a 24-hourcircadian pattern with peak serum levels in the early morning, 3-6 hoursafter onset of sleep, and nadir levels around midnight. Physical andpsychological stressors also activate cortisol secretion. Changedpatterns of serum cortisol levels have been observed in connection withabnormal adrenocorticotropic hormone (ACTH), levels, clinicaldepression, psychological stress, and physiological stressors such ashypoglycemia, illness, fever, trauma, surgery, fear, pain, physicalexertion, or temperature extremes. Cortisol levels and responsivenessmay also differ from normal for elderly individuals and in individualswith autism or Asperger's syndrome.

Glucocorticoids (GCs) such as, in humans, cortisol, perform severalimportant functions. These include participating in the regulation ofcarbohydrate, protein and fat metabolism by signaling the liver to makeglucose and glycogen, the adipose tissues to release lipids and fattyacids into the bloodstream, and the skeletal muscles to release proteinsor amino acids into the bloodstream. GCs also decrease bone formation.

GCs also regulate the body's inflammatory response as well. GCs are partof the feedback mechanism in the immune system that inhibits immuneactivity (i.e., inflammation). GCs cause their effects by binding to theGCR. The activated GCR complex in turn up-regulates the expression ofanti-inflammatory proteins in the nucleus (a process known astransactivation) and represses the expression of pro-inflammatoryproteins in the cytosol by preventing the translocation of othertranscription factors from the cytosol into the nucleus(transrepression) (Rhen T and Cidlowski J A. NEJM 2005; 353: 1711-23).

GCR antagonist therapy is helpful in patients with abnormally highlevels of cortisol (but maintained circadian rhythm), overresponsiveness to normal levels, or high night time cortisol levels as afeature of disrupted circadian rhythm. Successful therapeutic use ofsuch agents is thus dependent on determining circadian cortisol levels(either peak levels during the day, e.g., at noon, or measurements takenevery 4 hours or 6 hours over a 24 hour period). This combined system ofsalivary cortisol quantification as an enabling device for its pairedGCR antagonist will identify individuals for whom GCR antagonist therapyhas a benefit.

The glucocorticoid receptor (GR) is expressed at high levels in somenormal tissues, but not in others. Likewise, malignant tumors of diversetypes and sites have variable GR expression. When present in normal ortumor (benign or malignant) tissues, this GR expression may be variouslylocated in some or all of their cellular sub-compartments: 1. stemcells; 2. progenitor (so called “transit amplifying”) cell descendentsof activated stem cells; and 3. differentiated progeny of activated stemor progenitor cells.

As an example, in the gastrointestinal tract, GR are highly expressed inesophageal squamous epithelia, hepatocytes, and pancreatic islet cells,but are not highly expressed in other gastrointestinal epithelia(stomach, small and large intestines, pancreatic and biliary ducts). Incorresponding malignancies arising in these epithelia, hepatocellularcarcinoma (HCC) and squamous cell carcinomas (SCC) of the esophagus haveconsistently high GR expression. Gastric and colorectal adenocarcinomashave little to no GR expression.

Dexamethasone (DEX), a binding activator of GR, has been found to conferchemoresistance in oesophageal SCC and HCC cells, suggesting that GRexpression may be biologically important in some GR-expressingcarcinomas. This not only suggests why DEX or other glucocorticoids arenot useful in treatment of these malignancies, but it implies thatendogenous, circulating cortisol itself may actually promotechemoresistance, even in the absence of iatrogenic glucocorticoidadministration. Therefore, these findings suggest that blockade of GRwithin such malignant tumors, by preventing activation by endogenous,circulating cortisol, can play a role in maintaining or promotingchemosensitivity and/or treating neoplasia.

The present invention therefore relates to the use of GR antagonists(e.g., ORG 34517, RU486, and others) for the treatment of, for example,esophageal SCC and HCC or other tumors with high GR expression as ameans of inhibiting promotion of chemoresistance by endogenous cortisol.These effects may be present in all tumor cells or, when tumors havestem or progenitor cell compartments, these, specifically, as well.Thus, the present invention relates to the inhibition of chemopreventionin the bulk of cells making up a given tumor and/or in the rarestem/progenitor cells within the tumor that are often responsible fortumor resistance to therapy and re-occurrence, i.e., as a novel,targeted “cancer stem cell” treatment.

To avoid possible negative side effects of systemic blockade of GR, thepresent invention further relates to localized tumor treatment with GRantagonists through direct vascular infusion of tumor feeding vessels orby direct, intratumoral injection.

The present invention relates to the use of GR antagonists for thetreatment of, for example, breast and other cancers. The invention isbased on the observation that GR inhibition will increase tumor cellsusceptibility. GR antagonists will block anti-apoptotic GR signaling inGR-overexpressing breast cancer cells and subsequently render breastcancer cells more susceptible to conventional and novel cytotoxictherapies (via blocking GR's pro-cell survival signaling pathway).

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a method for detecting the presence ofa biological substance in a test sample, comprising the steps of:providing a test sample consisting of, for example, saliva, or a bodilyfluid sample from a subject with, for example, a lollipop-like apparatusincluding a stem integrated with the base and a head integrated with thestem. The stem head including a receptor of a sponge like carrier toensure a high void volume to absorb sufficient saliva, oral fluid or abodily fluid sample.

Combining the test sample with a buffering system (Reagent 1) containingviscosity controllers and stabilizers into a reaction vessel, mixing thesolution well, and expressing all the liquid from the sample carrierapparatus into Reagent 1 in the reaction vessel and discarding. Readingthe reaction vessel with sample and buffer for a fluorescencepolarization blank and then combining the test sample and buffer mixturewith a fluorescence-labeled ligand (Reagent 2) to said biologicalsubstance in the reaction vessel, mix solution well, to produce an assaysolution. Furthermore, Reagent 2 may be delivered to the reaction vesselwithout further dilution volume of the assay solution.

The invention provides a method for screening a patient for a diseasestate suitable for GCR (glucocorticoid receptor) antagonist therapy,comprising the steps of: a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; and h) comparing the patient's measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to thepredetermined reference range, then the patient has a disease statewhich involves elevated cortisol, and thus has a disease state which isa potential candidate for GCR antagonist therapy. The invention furtherprovides a method wherein the patient's test sample is selected from thegroup consisting of saliva, blood, plasma, serum, urine, other bodilyfluids, and combinations thereof. The invention further provides amethod wherein the sample is obtained from the patient over more thanone time, and the predetermined time is selected from the groupconsisting of morning, noon, and evening. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein themethod is to determine the circadian cycle of the cortisol levels in thepatient, and the predetermined time is selected from the groupconsisting of about hourly, every 4 hours, every 6 hours, every 8 hours,and every 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method further comprising thesteps of: i) determining a patient's elevated cortisol level; and j)providing a therapeutic for such elevated cortisol level, wherein thetherapeutic comprises GCR antagonist therapy. The invention furtherprovides a method wherein the patient has changed patterns of cortisollevels that have been observed in connection with abnormalAdrenocorticotropic hormone (ACTH) levels. The invention furtherprovides a method wherein the patient has non-normal cortisol levelsproduced by the adrenal cortex or disordered circadian rhythms, as amethod for selecting subjects for GCR antagonist therapy wherein thepatient has cortisol levels selected from the group consistingabnormally high cortisol levels but maintained circadian rhythm, overresponsiveness to normal levels, and high night time cortisol levels asa feature of disrupted circadian rhythm. The invention further providesa method wherein the disease state is selected from the group consistingof cancer, clinical depression, psychological stress, and physiologicalstressors such as hypoglycemia, illness, fever, trauma, surgery, fear,pain, physical exertion, or temperature extremes.

The invention provides a method for monitoring changes in cortisollevels in response to treatment, in patients who have non-normalcortisol levels produced by the adrenal cortex, comprising: a) obtaininga test sample from the patient, optionally at a predetermined time,using a test sample collection unit; b) combining said test sample witha buffering system to form a mixture in a reaction unit; c) measuring aparameter of the mixture to determine a blank measurement; d) combiningsaid test sample and buffer mixture with a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay solution; or combining said testsample and buffer mixture and delivering it to a carrier containing alabeled ligand which binds cortisol, wherein the labeled ligand isprovided in a label unit, in the reaction unit to produce an assayimmobilized complex; e) measuring a parameter of said assay solution orcomplex; f) comparing the measurement of the assay solution relative tothe blank measurement; g) determining the patient's circulating cortisollevels based on the change of the measurement; h) administering a GCRantagonist; i) repeating steps a) to f) after the therapy has beenadministered; and j) determining the patient's circulating cortisollevels post-therapy, wherein when the cortisol levels change in responseto treatment to indicate responsiveness to the GCR antagonist. Theinvention further provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionfurther provides a method wherein the sample is obtained from thepatient over more than one day, and the predetermined time is selectedfrom the group consisting of morning, noon, and evening. The inventionfurther provides a method wherein the sample is obtained from thepatient over consecutive days. The invention further provides a methodwherein the method is to determine the circadian cycle of the cortisollevels in the patient, and the predetermined time is selected from thegroup consisting of hourly, every 4 hours, every 6 hours, every 8 hours,and every 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans.

The invention provides a method for monitoring changes in cortisollevels in response to treatment and adjusting the treatment in responseto these changes in a patient who has non-normal cortisol levelsproduced by the adrenal cortex, comprising: a) obtaining a test samplefrom the patient, optionally at a predetermined time, using a testsample collection unit; b) combining said test sample with a bufferingsystem to form a mixture in a reaction unit; c) measuring a parameter ofthe mixture to determine a blank measurement; d) combining said testsample and buffer mixture with a labeled ligand which binds cortisol,wherein the labeled ligand is provided in a label unit, in the reactionunit to produce an assay solution; or combining said test sample andbuffer mixture and delivering it to a carrier containing a labeledligand which binds cortisol, wherein the labeled ligand is provided in alabel unit, in the reaction unit to produce an assay immobilizedcomplex; e) measuring a parameter of said assay solution or complex; f)comparing the measurement of the assay solution relative to the blankmeasurement; g) determining the patient's circulating cortisol levelsbased on the change of the measurement; h) administering a GCRantagonist; i) repeating steps a) to f) after the therapy has beenadministered; j) determining the patient's circulating cortisol levelspost-therapy; and k) adjusting the GCR antagonist therapy in response tochanges in the patient's cortisol levels post-therapy, wherein theadjustment in GCR antagonist therapy is to enhance therapeutic efficacy.

The invention further provides a method wherein the patient's testsample is selected from the group consisting of saliva, blood, plasma,serum, urine, other bodily fluids, and combinations thereof. Theinvention further provides a method wherein the sample is obtained fromthe patient over more than one time, and the predetermined time isselected from the group consisting of morning, noon, and evening. Theinvention further provides a method wherein the sample is obtained fromthe patient over consecutive days. The invention further provides amethod wherein the method is to determine the circadian cycle of thecortisol levels in the patient, and the predetermined time is selectedfrom the group consisting of hourly, every 4 hours, every 6 hours, every8 hours, and every 12 hours. The invention further provides a methodwherein the sample is obtained from the patient over consecutive days.The invention further provides a method wherein the predeterminedreference range is a medically standard reference range. The inventionfurther provides a method wherein the predetermined reference range isthe patient's previously measured level. The invention further providesa method wherein the ligand is detectably labeled with a moiety selectedfrom the group consisting of a radioisotope, a fluorophore, a quencherof fluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of compounds which areselective for GCR, compounds which non-specifically bind steroid hormonereceptors, and compounds which cross-react to both GCR and other steroidhormone receptors. The invention further provides a method wherein adecision to adjust the GCR antagonist therapy in response to changes inthe cortisol levels, post-therapy, is made by a medical professional.The invention further provides a method further comprising the step ofmonitoring changes in biomarker expression using a nucleic acidmicroarray. The invention further provides a method wherein in thepatients having normal baseline cortisol at the start of treatment, andchanging cortisol levels during treatment indicate responsiveness to theGCR antagonist. The invention further provides a method wherein thecombined system of salivary cortisol quantification as an enablingdevice for its paired GCR antagonist will identify patients for whom GCRantagonism has a likely benefit. The invention further provides a methodwherein the GCR antagonist is selected from the group consisting of ORG34517, 11-(substituted phenyl)-estra-4,9-diene derivatives, and11-(substituted phenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond. The invention furtherprovides a method wherein the buffering system comprises additionalcomponents selected form the group consisting of viscosity controllers,stabilizers, and combinations thereof. The invention further provides amethod wherein the fluorescence-labeled ligand which binds cortisol isselected from the group consisting of an aptamer, an antibody, anantibody fragment, a receptor, a receptor fragment, a bindingpolypeptide, a binding peptide, and combinations thereof. The inventionfurther provides a method wherein the test sample is collected from thepatient with a lollipop-like apparatus, including a stem integrated withthe base and a head integrated with the stem, and further wherein thestem head including a receptor of a sponge like carrier to ensure a highvoid volume to absorb sufficient sample.

The invention provides a method of treating major depressive disorder ina patient in need thereof by determining whether the patient has majordepressive disorder suitable for GCR (glucocorticoid receptor)antagonist therapy, comprising the steps of: a) obtaining a test samplefrom the patient, optionally at a predetermined time, using a testsample collection unit; b) combining said test sample with a bufferingsystem to form a mixture in a reaction unit; c) measuring a parameter ofthe mixture to determine a blank measurement; d) combining said testsample and buffer mixture with a labeled ligand which binds cortisol,wherein the labeled ligand is provided in a label unit, in the reactionunit to produce an assay solution; or combining said test sample andbuffer mixture and delivering it to a carrier containing a labeledligand which binds cortisol, wherein the labeled ligand is provided in alabel unit, in the reaction unit to produce an assay immobilizedcomplex; e) measuring a parameter of said assay solution or complex; f)comparing the measurement of the assay solution relative to the blankmeasurement; g) determining the patient's circulating cortisol levelsbased on the change of the measurement; h) comparing the measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to the apredetermined reference range, then the patient has major depressivedisorder which involves elevated cortisol, and thus has major depressivedisorder suitable for GCR (glucocorticoid receptor) antagonist therapy;and i) when the patient has major depressive disorder suitable for GCRantagonist therapy, a administering at least one GCR antagonist. Theinvention further provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionfurther provides a method wherein the sample is obtained from thepatient over more than one time, and the predetermined time is selectedfrom the group consisting of morning, noon, and evening. The inventionfurther provides a method wherein the sample is obtained from thepatient over consecutive days. The invention further provides a methodwherein the method is to determine the circadian cycle of the cortisollevels in the patient, and the predetermined time is selected from thegroup consisting of hourly, every 4 hours, every 6 hours, every 8 hours,and every 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCR(glucocorticoid receptor) antagonist is selected from the groupconsisting of ORG 34517, 11-(substituted phenyl)-estra-4,9-dienederivatives, and 11-(substituted phenyl)-estra-4,9-diene derivatives offormula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a method of treating psychotic depression in apatient in need thereof by determining whether the patient has psychoticdepression suitable for GCR (glucocorticoid receptor) antagonisttherapy, comprising the steps of: a) obtaining a test sample from thepatient, optionally at a predetermined time using a test samplecollection unit; b) combining said test sample with a buffering systemto form a mixture in a reaction unit; c) measuring a parameter of themixture to determine a blank measurement; d) combining said test sampleand buffer mixture with a labeled ligand which binds cortisol, whereinthe labeled ligand is provided in a label unit, in the reaction unit toproduce an assay solution; or combining said test sample and buffermixture and delivering it to a carrier containing a labeled ligand whichbinds cortisol, wherein the labeled ligand is provided in a label unit,in the reaction unit to produce an assay immobilized complex; e)measuring a parameter of said assay solution or complex; f) comparingthe measurement of the assay solution relative to the blank measurement;g) determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the predetermined reference range,then the patient has psychotic depression which involves elevatedcortisol, and thus has psychotic depression suitable for GCR antagonisttherapy; and i) when the patient has psychotic depression suitable forGCR antagonist therapy, administering at least one GCR antagonist. Theinvention further provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionfurther provides a method wherein the sample is obtained from thepatient over more than one time, and the predetermined time is selectedfrom the group consisting of morning, noon, and evening. The inventionfurther provides a method wherein the sample is obtained from thepatient over consecutive days. The invention further provides a methodwherein the method is to determine the circadian cycle of the cortisollevels in the patient, and the predetermined time is selected from thegroup consisting of hourly, every 4 hours, every 6 hours, every 8 hours,and every 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of ORG 34517,11-(substituted phenyl)-estra-4,9-diene derivatives, and 11-(substitutedphenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a method of treating stress-induced cortisolelevation in a patient in need thereof by determining whether thepatient has stress-induced cortisol elevation suitable for GCR(glucocorticoid receptor) antagonist therapy, comprising the steps of:a) obtaining a test sample from the patient, optionally at apredetermined time, using a test sample collection unit; b) combiningsaid test sample with a buffering system to form a mixture in a reactionunit; c) measuring a parameter of the mixture to determine a blankmeasurement; d) combining said test sample and buffer mixture with alabeled ligand which binds cortisol, wherein the labeled ligand isprovided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex;

f) comparing the measurement of the assay solution relative to the blankmeasurement; g) determining the patient's circulating cortisol levelsbased on the change of the measurement; h) comparing the measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to thepredetermined reference range, then the patient has stress-inducedcortisol elevation which involves elevated cortisol, and thus hasstress-induced cortisol elevation suitable for GCR antagonist therapy;and i) when the patient has stress-induced cortisol elevation suitablefor GCR antagonist therapy, administering at least one GCR antagonist.The invention further provides a method wherein the patient's testsample is selected from the group consisting of saliva, blood, plasma,serum, urine, other bodily fluids, and combinations thereof. Theinvention further provides a method wherein the stress-related cortisolelevation is related to a hospital stay, medical treatment, aninstitutional stay, clinical depression, psychological stress,physiological stressors, hypoglycemia, illness, fever, trauma, surgery,fear, pain, physical exertion, or temperature extremes. The inventionfurther provides a method wherein the patient is an elderly individual.The invention further provides a method wherein the patient has autismor Asperger's syndrome. The invention further provides a method whereinthe sample is obtained from the patient over more than one time, and thepredetermined time is selected from the group consisting of morning,noon, and evening. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the method is to determine thecircadian cycle of the cortisol levels in the patient, and thepredetermined time is selected from the group consisting of hourly,every 4 hours, every 6 hours, every 8 hours, and every 12 hours. Theinvention further provides a method wherein the sample is obtained fromthe patient over consecutive days. The invention further provides amethod wherein the predetermined reference range is a medically standardreference range. The invention further provides a method wherein thepredetermined reference range is the patient's previously measuredlevel. The invention further provides a method wherein the ligand isdetectably labeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, anaffinity tag, and an epitope tag. The invention further provides amethod wherein said measuring of said parameter of said mixture and saidassay solution is performed using a method selected from spectroscopic,photochemical, radiochemical, biochemical, enzymatic, immunochemical,chemical label displacement, surface plasmon resonance, fluorescenceresonance energy transfer, fluorescence quenching, lateral flow, andfluorescence polarization means. The invention further provides a methodwherein the GCR antagonist is selected from the group consisting of ORG34517, 11-(substituted phenyl)-estra-4,9-diene derivatives, and11-(substituted phenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a method of treating post-traumatic stressdisorder in a patient in need thereof by determining whether the patienthas post-traumatic stress disorder suitable for GCR (glucocorticoidreceptor) antagonist therapy, comprising the steps of: a) obtaining atest sample from the patient, optionally at a predetermined time, usinga test sample collection unit; b) combining said test sample with abuffering system to form a mixture in a reaction unit; c) measuring aparameter of the mixture to determine a blank measurement; d) combiningsaid test sample and buffer mixture with a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay solution; or combining said testsample and buffer mixture and delivering it to a carrier containing alabeled ligand which binds cortisol, wherein the labeled ligand isprovided in a label unit, in the reaction unit to produce an assayimmobilized complex; e) measuring a parameter of said assay solution orcomplex; f) comparing the measurement of the assay solution relative tothe blank measurement; g) determining the patient's circulating cortisollevels based on the change of the measurement; h) comparing the measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to thepredetermined reference range, then the patient has post-traumaticstress disorder which involves elevated cortisol, and thus haspost-traumatic stress disorder suitable for GCR (glucocorticoidreceptor) antagonist therapy; and i) when the patient has post-traumaticstress disorder suitable for GCR antagonist therapy, administering atleast one GCR antagonist. The invention further provides a methodwherein the patient's test sample is selected from the group consistingof saliva, blood, plasma, serum, urine, other bodily fluids, andcombinations thereof. The invention further provides a method whereinthe sample is obtained from the patient over more than one time, and thepredetermined time is selected from the group consisting of morning,noon, and evening. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the method is to determine thecircadian cycle of the cortisol levels in the patient, and thepredetermined time is selected from the group consisting of hourly,every 4 hours, every 6 hours, every 8 hours, and every 12 hours. Theinvention further provides a method wherein the sample is obtained fromthe patient over consecutive days. The invention further provides amethod wherein the predetermined reference range is a medically standardreference range. The invention further provides a method wherein thepredetermined reference range is the patient's previously measuredlevel. The invention further provides a method wherein the ligand isdetectably labeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, anaffinity tag, and an epitope tag. The invention further provides amethod wherein said measuring of said parameter of said mixture and saidassay solution is performed using a method selected from spectroscopic,photochemical, radiochemical, biochemical, enzymatic, immunochemical,chemical label displacement, surface plasmon resonance, fluorescenceresonance energy transfer, fluorescence quenching, lateral flow, andfluorescence polarization means. The invention further provides a methodwherein the GCR antagonist is selected from the group consisting of ORG34517, 11-(substituted phenyl)-estra-4,9-diene derivatives, and11-(substituted phenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a method of prevention of weight gain in patientsusing anti-psychotic or anti-depressant medications in a patient in needthereof, wherein the weight gain is suitable for GCR (glucocorticoidreceptor) antagonist therapy, comprising the steps of: a) obtaining atest sample from the patient, optionally at a predetermined time using atest sample collection unit; b) combining said test sample with abuffering system to form a mixture in a reaction unit; c) measuring aparameter of the mixture to determine a blank measurement; d) combiningsaid test sample and buffer mixture with a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay solution; or combining said testsample and buffer mixture and delivering it to a carrier containing alabeled ligand which binds cortisol, wherein the labeled ligand isprovided in a label unit, in the reaction unit to produce an assayimmobilized complex; e) measuring a parameter of said assay solution orcomplex;

f) comparing the measurement of the assay solution relative to the blankmeasurement; g) determining the patient's circulating cortisol levelsbased on the change of the measurement; h) comparing the measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to thepredetermined reference range, then the patient has weight gain whichinvolves elevated cortisol, which is suitable for GCR (glucocorticoidreceptor) antagonist therapy; and i) when the patient has weight gainsuitable for GCR antagonist therapy, administering at least one GCRantagonist. The invention further provides a method wherein thepatient's test sample is selected from the group consisting of saliva,blood, plasma, serum, urine, other bodily fluids, and combinationsthereof. The invention further provides a method wherein the sample isobtained from the patient over more than one time, and the predeterminedtime is selected from the group consisting of morning, noon, andevening. The invention further provides a method wherein the sample isobtained from the patient over consecutive days. The invention furtherprovides a method wherein the method is to determine the circadian cycleof the cortisol levels in the patient, and the predetermined time isselected from the group consisting of hourly, every 4 hours, every 6hours, every 8 hours, and every 12 hours. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein thepredetermined reference range is a medically standard reference range.The invention further provides a method wherein the predeterminedreference range is the patient's previously measured level. Theinvention further provides a method wherein the ligand is detectablylabeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, anaffinity tag, and an epitope tag. The invention further provides amethod wherein said measuring of said parameter of said mixture and saidassay solution is performed using a method selected from spectroscopic,photochemical, radiochemical, biochemical, enzymatic, immunochemical,chemical label displacement, surface plasmon resonance, fluorescenceresonance energy transfer, fluorescence quenching, lateral flow, andfluorescence polarization means. The invention further provides a methodwherein the GCR antagonist is selected from the group consisting of ORG34517, 11-(substituted phenyl)-estra-4,9-diene derivatives, and11-(substituted phenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a method of treating Cushing's syndrome in apatient in need thereof by determining whether the patient has Cushing'ssyndrome suitable for GCR (glucocorticoid receptor) antagonist therapy,comprising the steps of: a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the predetermined reference range,then the patient has Cushing's syndrome which involves elevatedcortisol, which is suitable for GCR (glucocorticoid receptor) antagonisttherapy; and i) when the patient has Cushing's syndrome suitable for GCRantagonist therapy, administering at least one GCR antagonist. Theinvention further provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionfurther provides a method wherein the sample is obtained from thepatient over more than one time, and the predetermined time is selectedfrom the group consisting of morning, noon, and evening. The inventionfurther provides a method wherein the sample is obtained from thepatient over consecutive days. The invention further provides a methodwherein the method is to determine the circadian cycle of the cortisollevels in the patient, and the predetermined time is selected from thegroup consisting of hourly, every 4 hours, every 6 hours, every 8 hours,and every 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of ORG 34517,11-(substituted phenyl)-estra-4,9-diene derivatives, and 11-(substitutedphenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a pharmaceutical composition comprising: (a) GCR(glucocorticoid receptor) antagonist; (b) the pharmaceutical compositionof (a), further comprising at least one pharmaceutically acceptableexcipient; (c) the pharmaceutical composition of (a) or (b), wherein thepharmaceutical composition is formulated or manufactured as a liquid, anelixir, an aerosol, a spray, a powder, a tablet, a pill, a capsule, agel, a geltab, a nanosuspension, a nanoparticle, an extended releasedosage form, or a topical formulation. The invention provides a methodfor treating a condition selected from the group consisting of majordepressive disorder, psychotic depression, stress-induced cortisolelevation, post-traumatic stress disorder, preventing weight gain inpatients using anti-psychotic and anti-depressant medications, or havingCushing's syndrome, in a patient in need of such treatment comprisingadministering the pharmaceutical composition to the patient.

The invention provides a kit for the treatment, amelioration orprevention of a condition selected from the group consisting of majordepressive disorder, psychotic depression, stress-induced cortisolelevation, post-traumatic stress disorder, preventing weight gain inpatients using anti-psychotic and anti-depressant medications, or havingCushing's syndrome, in a patient in need of such treatment comprising:(a) the pharmaceutical composition; and (b) at least one blisterpackage; a lidded blister; a blister card or packet; a clamshell; anintravenous (IV) package, IV packette or IV container; a tray or ashrink wrap comprising the pharmaceutical composition of (a) andinstructions for use of the pharmaceutical composition.

The invention provides a product of manufacture comprising a blisterpackage; a lidded blister; a blister card or packet; a clamshell; anintravenous (IV) package, IV packette or IV container; a tray or ashrink wrap comprising the pharmaceutical composition and instructionsfor use of the pharmaceutical composition. The invention furtherprovides a method wherein the GCR (glucocorticoid receptor) antagonistselected from the group consisting of ORG 34517, 11-(substitutedphenyl)-estra-4,9-diene derivatives, and 11-(substitutedphenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond.

The invention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor, in a patient in need of suchtreatment, comprising: administering to said animal or humantherapeutically effective amounts of each of at least oneneoplasia-treating agent and a GCR (glucocorticoid receptor) antagonistselected from the group consisting of ORG 34517, 11-(substitutedphenyl)-estra-4,9-diene derivatives, and 11-(substitutedphenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond. The invention furtherprovides a method wherein ORG34517 is administered as an agent whichdirectly effects tumor growth, independent of other administeredtreatment modalities, for palliation, remission, or cure. The inventionfurther provides a method wherein said neoplasia treating agent isradiation. The invention further provides a method wherein saidneoplasia treating agent is a biotherapy agent. The invention furtherprovides a method wherein said neoplasia treating agent is achemotherapy agent. The invention further provides a method wherein saidneoplasia treating agent is a radionuclide. The invention furtherprovides a method wherein the neoplasia is selected for the groupconsisting of hepatocellular carcinoma, esophageal squamous cellcarcinoma, breast cancer, pancreatic cancer, squamous cell cancer oradenocarcinoma of the head and neck, colorectal cancer, renal cancer,brain cancer, prostate cancer, small and non-small cell lung cancer,bladder cancer, bone or joint cancer, uterine cancer, cervical cancer,multiple myeloma, hematopoietic malignancies, lymphoma, Hodgkin'sdisease, non-Hodgkin's lymphoma, skin cancer, melanoma, squamous cellcarcinoma, leukemia, lung cancer, ovarian cancer, stomach cancer,Kaposi's sarcoma, laryngeal cancer, endocrine carcinomas, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the pituitarygland, cancer of the adrenal gland, and combinations thereof. Theinvention further provides a method wherein the neoplasia expressesmultidrug resistance genes when GR is activated through binding byendogenous cortisol. The invention further provides a method wherein theneoplasia expresses proteins of cell survival pathway (includinginhibition of apoptosis) genes when GR is activated through binding byendogenous cortisol. The invention further provides a method wherein theneoplasia expresses genes responsible for epithelial-mesenchymaltransition and cell shape maintenance are repressed when GR is activatedthrough binding by endogenous cortisol. The invention further provides amethod wherein the neoplasia expresses genes involved in signaltransduction pathways, lipid/fatty acid metabolism, inflammation andmacrophage regulation, transcriptional regulation and chromatinremodeling, and cell metabolic pathways. The invention further providesa method wherein tumor stem cells (TSC) express GR, blockade of which byORG34517 results in anti-TSC therapy. The invention further provides amethod wherein TSC express multidrug resistance genes when GR isactivated through binding by endogenous cortisol. The invention furtherprovides a method wherein TSC express proteins of cell survival pathways(including inhibition of apoptosis) genes when GR is activated throughbinding by endogenous cortisol. The invention further provides a methodwherein TSC express genes responsible for epithelial-mesenchymaltransition and cell shape maintenance are repressed when GR is activatedthrough binding by endogenous cortisol. The invention further provides amethod wherein TSC express genes involved in signal transductionpathways, lipid/fatty acid metabolism, inflammation and macrophageregulation, transcriptional regulation and chromatin remodeling, andcell metabolic pathways. The invention further provides a method whereinthe neoplasia is chemo-resistant ER/GR+ breast cancer. The inventionfurther provides a method wherein the administration of ORG 34517 forGR-blockade, reduces toxicities and side effects when givensystemically. The invention further provides a method wherein the ORG34517 given systemically through oral or intravenous routes. Theinvention further provides a method wherein the ORG 34517 is targeted totumor by intra-arterial infusion to reduce systemic side effects of GRblockade. The invention further provides a method wherein the ORG 34517is given to accomplish cure or remission of tumor. The invention furtherprovides a method wherein the ORG 34517 is given to accomplish reductionof tumor burden to enhance effectiveness of subsequent surgicalresection. The invention further provides a method wherein the ORG 34517is given to accomplish reduction of tumor burden to make an unresectabletumor resectable.

The invention provides a method for treatment of neoplasia in a patientcomprising targeted delivery of ORG 34517, wherein the neoplasiaexpresses multidrug resistance genes when GR is activated throughbinding by endogenous cortisol. The invention further provides a methodwherein the HCC patient is not a candidate to undergo surgicalintervention because tumor is too large or encroaches on liver anatomyin a manner that prevents resection, delivery of ORG34517 prior toablative or chemotherapy to shrink the tumor and make it resectable. Theinvention further provides a method wherein the HCC is present incirrhosis and the patient is not a candidate for transplantation becauseof large tumor size, administration of ORG34517 making the tumoramenable to ablative or chemotherapy to shrink the tumor and allow thepatient to be eligible for liver transplant. The invention furtherprovides a method wherein the HCC is present in cirrhosis and thepatient is a candidate for transplantation, administration of ORG34517making the tumor amenable to ablative therapy to manage tumor whilepatient remains on the liver transplant waitlist.

The invention provides a method for low toxicity chemoprevention bytargeted liver infusion in patients with forms of established cirrhosisthat are high risk for emergence of HCC, including those withpremalignant lesions diagnosed on biopsy or by radiology, comprisingtargeted delivery of Org34517 to intrahepatic lesions, wherein thetargeted delivery of ORG g34517 to intrahepatic lesions preventsemergence of HCC. The invention further provides a method wherein thepatient with HCC is not a candidate for undergo surgical intervention.The invention further provides a method wherein the HCC resides inlocations where surgical or ablative interventions are not available.The invention further provides a method wherein the patient with HCC hascirrhosis that is too advanced to make partial hepatectomy safe. Theinvention further provides a method wherein the patient with HCC is tooearly in their chronic liver disease to qualify for transplantation. Theinvention further provides a method wherein the HCC is too advanced forlocalized treatments.

The invention provides a method for treatment of HCC comprising: a)targeted delivery of ORG 34517 to intrahepatic lesions, wherein thetargeted delivery of ORG 34517 to intrahepatic lesions improves outcomesof localized chemo-ablative therapies. The invention further provides amethod wherein the treatment is to help patients qualify for livertransplantation.

The invention provides a method for low toxicity chemoprevention bytargeted liver infusion in patients with forms of established cirrhosisthat are high risk for emergence of HCC, including those withpremalignant lesions diagnosed on biopsy or by radiology comprisingtargeted delivery of ORG 34517 to intrahepatic lesions, wherein thetargeted delivery of ORG 34517 to intrahepatic lesions preventsemergence of HCC. The invention further provides a method wherein theneoplasia is eSCC.

The invention provides a method for treatment of eSCC in a patient withunresectable eSCC where systemic or targeted administration of ORG 34517makes tumor responsive to ablative or chemotherapies as palliative orcurative treatment.

The invention provides method for treatment of eSCC in a patient withunresectable eSCC where systemic or targeted administration of ORG 34517makes tumor responsive to ablative or chemotherapies to shrink the tumorand enhance resectability. The invention further provides a methodwherein the neoplasia-treating agent is a chemotherapeutic agentincluding but not limited to gemcitabine, paclitaxel, carboplatin,cisplatin, and 5-fluorouracil. The invention further provides a methodwherein the therapeutic effective amount of glucocorticoid administeredis about 100 to 400 microg/kg body weight per day when administeredintravenously.

The invention provides a method for treating neoplasia, in an animal orhuman in need of such treatment, wherein said neoplasia comprisesneoplastic stem cells characterized by expression of a glucocorticoidreceptor, and further characterized by expression of multidrugresistance genes or other stem cell related means of survival when GR isactivated through binding by endogenous cortisol, the method comprising:a) administering to said animal or human a therapeutically effectiveamount of ORG 34517; and b) administering to said animal or human atherapeutically effective amount of the at least one neoplasia-treatingagent, wherein said therapeutically effective amount of ORG 34517 is anamount sufficient to promote susceptibility of the neoplastic stem cellsto at least one neoplasia-treating agent. The invention further providesa method wherein said neoplasia-treating agent is radiation selectedfrom the group consisting of external beam or radionuclide therapy. Theinvention further provides a method wherein said neoplasia-treatingagent is a biotherapy agent. The invention further provides a methodwherein said neoplasia-treating agent is a chemotherapy agent. Theinvention further provides a method wherein said neoplasia-treatingagent is a radionuclide. The invention further provides a method whereinthe neoplasia is selected from the group consisting of hepatocellularcarcinoma, esophageal squamous cell carcinoma, breast cancer, pancreaticcancer, squamous cell cancer or adenocarcinoma of the head and neck,colorectal cancer, renal cancer, brain cancer, prostate cancer, smalland non-small cell lung cancer, bladder cancer, bone or joint cancer,uterine cancer, cervical cancer, multiple myeloma, hematopoieticmalignancies, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skincancer, melanoma, squamous cell carcinoma, leukemia, lung cancer,ovarian cancer, stomach cancer, Kaposi's sarcoma, laryngeal cancer,endocrine carcinomas, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the pituitary gland, cancer of the adrenalgland, and combinations thereof. The invention further provides a methodwherein the neoplasia expresses multidrug resistance genes when GR isactivated through binding by endogenous cortisol.

The invention provides a pharmaceutical composition for treatingneoplasia in a patient which is characterized by expression of aglucocorticoid receptor, comprising: a) therapeutically effectiveamounts of at least one neoplasia-treating agent; b) a GCR(glucocorticoid receptor) antagonist selected from the group consistingof ORG 34517, 11-(substituted phenyl)-estra-4,9-diene derivatives, and11-(substituted phenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), O, and NOH; andthe interrupted line represents an optional bond; and c) optionally, atleast one pharmaceutically acceptable carrier. The invention furtherprovides a method wherein said neoplasia-treating agent is selected fromthe group consisting of a chemotherapeutic agent, a biotherapeuticagent, a radionuclide agent, and combinations thereof. The inventionfurther provides a method wherein the neoplasia is selected from thegroup consisting of hepatocellular carcinoma, esophageal squamous cellcarcinoma, breast cancer, pancreatic cancer, squamous cell cancer oradenocarcinoma of the head and neck, colorectal cancer, renal cancer,brain cancer, prostate cancer, small and non-small cell lung cancer,bladder cancer, bone or joint cancer, uterine cancer, cervical cancer,multiple myeloma, hematopoietic malignancies, lymphoma, Hodgkin'sdisease, non-Hodgkin's lymphoma, skin cancer, melanoma, squamous cellcarcinoma, leukemia, lung cancer, ovarian cancer, stomach cancer,Kaposi's sarcoma, laryngeal cancer, endocrine carcinomas, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the pituitarygland, cancer of the adrenal gland, and combinations thereof. Theinvention further provides a method, wherein the neoplasia ischemo-resistant ER-GR+ breast cancer. The invention further provides amethod wherein the neoplasia expresses multidrug resistance genes whenGR is activated through binding by endogenous cortisol. The inventionfurther provides a method wherein the chemotherapeutic agent is selectedfrom the group comprising: busulfan, cisplatin, carboplatin,chlorambucil, cyclophosphamide, ifosfamide, dacarbazine (DTIC),mechlorethamine (nitrogen mustard), melphalan carmustine (BCNU)lomustine (CCNU), 5-FU, capecitabine, methotrexate, gemcitabine,cytarabine (ara-C), fludarabine dactinomycin, daunorubicin, doxorubicin(Adriamycin), idarubicin, mitoxantrone, paclitaxel, docetaxel, etoposide(VP-16), vinblastine, vincristine, vinorelbine prednisone,dexamethasone, tamoxifen, fulvestrant, anastrozole, letrozole, megestrolacetate, bicalutamide, flutamide, leuprolide, goserelin, L-asparaginase,and tretinoin, gemcitabine, paclitaxel, carboplatin, 5-FU, andcombinations thereof.

The invention provides a diagnostic kit comprises the followingcomponents: a) test sample collection unit; b) the buffer system unit;c) the reaction unit; and d) the label unit, wherein the components arein a blister package; a lidded blister; a blister card or packet; aclamshell; a tray, or a shrink wrap, and instructions for use of thekit. The invention further provides test sample collection unit whereinthe test sample collection unit comprises a stem integrated with a base,and a head integrated with the stem. The invention further provides atest sample collection unit wherein the stem head comprises a receptorof a sponge-like carrier sufficient to ensure a high void volume toabsorb sufficient saliva, oral fluid or a bodily fluid sample. Theinvention further provides a buffer system unit wherein the buffersystem unit comprises additional components selected from the groupconsisting of viscosity controllers, stabilizers, and combinationsthereof. The invention further provides a reaction unit wherein thereaction unit is adapted to fit in a fluorescent polarization reader.The invention further provides a label unit wherein the label unitcomprises a fluorescence-labeled ligand which binds cortisol, whereinthe fluorescence-labeled ligand which binds cortisol is selected fromthe group consisting of an aptamer, an antibody, an antibody fragment, areceptor, a receptor fragment, a binding polypeptide, a binding peptide,and combinations thereof. The invention further provides a diagnostickit wherein the reader apparatus provides temperature control andon-board mixing as an aid in viscosity control of the reaction to ensurebetter accuracy and precision. The invention further provides adiagnostic kit wherein the reader is a miniaturized, portable apparatusfor measuring fluorescence polarization of a liquid sample by direct orindirect methods. The invention further provides a diagnostic kitwherein system also enables continual monitoring of the patient duringtreatment for assessment of responsiveness to treatment.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIG. 1 is an example of a sample carrier.

FIG. 2 is an example of glass reaction vessel with reagent one dispensedinside.

FIG. 3 is an example of a fluorescent ligand reagent two, dryformulation to be dissolved in the reaction vessel.

FIGS. 4A through 4D show examples of additional embodiments of thechemistry process configuration. FIG. 4A shows a blister pack of reagentcartridge units. FIG. 4B shows an exemplary reaction vessel and cap.FIG. 4C shows an exemplary sample carrier.

FIG. 4D shows an exemplary reagent 2 (fluorescent ligand) carrier.

FIGS. 5A through 5C show examples of additional embodiments of thechemistry process configuration. FIG. 5A shows an exemplary samplecarrier. FIG. 5B shows an exemplary reaction vessel and cap. FIG. 5Cshows an exemplary reagent 2 (fluorescent ligand) carrier.

FIGS. 6A and 6B show additional embodiments of chemistry processconfiguration-plastic cartridge. FIG. 6A shows an exemplary samplecarrier. FIG. 6B shows an exemplary reagent 2 (fluorescent ligand)carrier.

FIG. 7 is an example of a Fluorescence Polarization Reader (DC andbattery operated wt. <3 lbs).

FIG. 8 is Effect of Vehicle (Ethanol/Castor Oil)/Vehicle (Ethanol/SesameOil), Vehicle (Ethanol/Castor Oil)/Taxol (10 mg/kg/day) and ORG 34517(20.5 mg/kg/day)/Taxol (10 mg/kg/day) on relative tumor growth.Compounds are administered day 1-5.

FIG. 9 shows the Effect of Vehicle (Ethanol/Castor Oil)/Taxol (10mg/kg/day) and ORG 34517 (20.5 mg/kg/day)/Taxol (10 mg/kg/day) onrelative tumor growth. The average curve for Veh/Taxo is shown.517/Taxocurves are show for each individual animal Compounds areadministered day 1-5.

FIG. 10 shows the results of an experiment in which mice were implantedwith cultured ER-GR+ human breast cancer cells. As tumor volume in eachmouse approached the test threshold of 200 mm3, the mice were randomizedto receive intraperitoneal injections of vehicle alone, chemotherapy(Paclitaxel) alone, and chemotherapy and ORG 34517. Each group contained3 mice. Results show significant differences in attained tumor volume

FIG. 11 shows the AUC₀₋₂₄. Two dogs were included in the test. Thenanosuspension increases the exposure of(11b,17b)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a low cost rapid response diagnostic system todetermine salivary cortisol levels in patients selected as potentialcandidates for GCR (glucocorticoid receptor) antagonist therapyutilizing a GCR antagonist, such as ORG 34517. The rapid, sensitive, andinexpensive test can be used to determine patients who have non-normalcortisol production or disordered circadian rhythms as a method forselecting subjects for GCR antagonist therapy for whom it is likely tohave beneficial and/or therapeutic effects, and can also be used tomonitor changes in cortisol levels in response to treatment.

As used herein, the term “agent” refers to any molecule, compound,methodology and/or substance for use in the prevention, treatment,management and/or diagnosis of cancer. Non-limiting examples of cancertherapies include chemotherapies, radiation therapies, hormonaltherapies, anti-angiogenesis therapies, targeted therapies, and/orbiological therapies including immunotherapies and surgery

As used herein, the term “cancer cells” refer to cells that acquire acharacteristic set of functional capabilities during their development,including the ability to evade apoptosis, self-sufficiency in growthsignals, insensitivity to anti-growth signals, tissueinvasion/metastasis, significant growth potential, and/or sustainedangiogenesis. The term “cancer cell” is meant to encompass bothpre-malignant and malignant cancer cells.

As used herein, the term “cancer stem cell(s)” refers to a cell that canbe a progenitor of a highly proliferative cancer cell. A cancer stemcell has the ability to re-grow a tumor as demonstrated by its abilityto form tumors in immunocompromised mice, and typically to form tumorsupon subsequent serial transplantation in immunocompromised mice. Cancerstem cells are also typically slow-growing relative to the bulk of atumor; that is, cancer stem cells are generally quiescent. In certainembodiments, but not all, the cancer stem cell may representapproximately 0.1 to 10% of a tumor.

As used herein, the phrase “diagnostic agent” refers to any molecule,compound, and/or substance that is used for the purpose of diagnosing adisease or condition. Non-limiting examples of diagnostic agents includeantibodies, antibody fragments, or other proteins, including thoseconjugated to a detectable agent. As used herein, the term “detectableagents” refer to any molecule, compound and/or substance that isdetectable by any methodology available to one of skill in the art.Non-limiting examples of detectable agents include dyes, gases, metals,or radioisotopes. As used herein, diagnostic agent and “imaging agent”are equivalent terms.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of a disease or condition, such ascancer, and one or more symptoms thereof, to enhance or improve theprophylactic effect(s) of another therapy, reduce the severity, theduration of a disease or condition, such as cancer, ameliorate one ormore symptoms of a disease or condition, such as cancer, prevent theadvancement of a disease or condition, such as cancer, cause regressionof a disease or condition, such as cancer, and/or enhance or improve thetherapeutic effect(s) of another therapy.

In an embodiment of the invention, the amount of a therapy is effectiveto achieve one, two, three, or more results following the administrationof one, two, three or more therapies: (1) a stabilization, reduction orelimination of the cancer stem cell population; (2) a stabilization,reduction or elimination in the cancer cell population; (3) astabilization or reduction in the growth of a tumor or neoplasm; (4) animpairment in the formation of a tumor; (5) eradication, removal, orcontrol of primary, regional and/or metastatic cancer; (6) a reductionin mortality; (7) an increase in disease-free, relapse-free,progression-free, and/or overall survival, duration, or rate; (8) anincrease in the response rate, the durability of response, or number ofpatients who respond or are in remission; (9) a decrease inhospitalization rate; (10) a decrease in hospitalization lengths; (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%; (12) an increase in the number of patients inremission; (13) an increase in the length or duration of remission; (14)a decrease in the recurrence rate of cancer; (15) an increase in thetime to recurrence of cancer; and (16) an amelioration of cancer-relatedsymptoms and/or quality of life.

As used herein, the phrase “pharmaceutically acceptable” means approvedby a regulatory agency of the federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopeia, or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans.

As used herein, the term “predetermined reference range” refers to areference range for the particular biological entity, e.g., cortisol,for a subject or a population of subjects. Each laboratory may establishits own reference range for each particular assay, or a standardreference range for each assay may be made available and used locally,regionally, nationally, or worldwide or may be patient-specific. In onespecific embodiment, the term refers to a reference range for the amountof cortisol in a patient or a specimen from a patient. In anotherspecific embodiment, the term refers to a reference range for the amountof cortisol in a patient or a specimen from a patient.

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy to a subject refer to theprevention or inhibition of the recurrence, onset, and/or development ofa a disease or condition, such as cancer or a symptom thereof in asubject resulting from the administration of a therapy (e.g., aprophylactic or therapeutic agent), or a combination of therapies (e.g.,a combination of prophylactic or therapeutic agents). In someembodiments, such terms refer to one, two, three, or more resultsfollowing the administration of one or more therapies: (1) astabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) an increase in response rate; (4) an increase inthe length or duration of remission; (5) a decrease in the recurrencerate of cancer; (6) an increase in the time to recurrence of cancer; (7)an increase in the disease-free, relapse-free, progression-free, and/oroverall survival of the patient; and (8) an amelioration ofcancer-related symptoms and/or quality of life. In specific embodiments,such terms refer to a stabilization, reduction or elimination of thecancer stem cell population.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the term “patient” refers to an animal,preferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), and mostpreferably a human. In some embodiments, the subject is a non-humananimal such as a farm animal (e.g., a horse, pig, or cow) or a pet(e.g., a dog or cat). In a specific embodiment, the subject is anelderly human. In another embodiment, the subject is a human adult. Inanother embodiment, the subject is a human child. In yet anotherembodiment, the subject is a human infant.

As used herein, the term “therapeutic agent” refers to any molecule,compound, and/or substance that is used for the purpose of treatingand/or managing a disease or disorder. Examples of therapeutic agentsinclude, but are not limited to, proteins, immunoglobulins (e.g.,multi-specific Igs, single chain Igs, Ig fragments, polyclonalantibodies and their fragments, monoclonal antibodies and theirfragments), peptides (e.g., peptide receptors, selectins), bindingproteins, biologics, chemospecific agents, chemotoxic agents (e.g.,anti-cancer agents), proliferation-based therapy, radiation,chemotherapy, anti-angiogenic agents, and small molecule drugs.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a disease or condition, suchas cancer, or one or more symptoms thereof. In certain embodiments, theterms “therapy” and “therapies” refer to chemotherapy, small moleculetherapy, radioimmunotherapy, toxin therapy, prodrug-activating enzymetherapy, biologic therapy, antibody therapy, surgical therapy, hormonetherapy, immunotherapy, anti-angiogenic therapy, targeted therapy,epigenetic therapy, demethylation therapy, histone deacetylase inhibitortherapy, differentiation therapy, radiation therapy, or a combination ofthe foregoing and/or other therapies useful in the prevention,management and/or treatment of a cancer or one or more symptoms thereof.

As used herein, the terms “treat,” “treatment,” and “treating” in thecontext of the administration of a therapy to a subject refer to thereduction or inhibition of the progression and/or duration of a diseaseor condition, such as cancer, the reduction or amelioration of theseverity of a disease or condition, such as cancer, and/or theamelioration of one or more symptoms thereof resulting from theadministration of one or more therapies. In specific embodiments, suchterms refer to one, two or three or more results following theadministration of one, two, three or more therapies: (1) astabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) a stabilization or reduction in the growth of atumor or neoplasm; (4) an impairment in the formation of a tumor; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalization rate; (10) a decrease in hospitalization lengths; (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%; and (12) an increase in the number of patientsin remission. In certain embodiments, such terms refer to astabilization or reduction in the cancer stem cell population. In someembodiments, such terms refer to a stabilization or reduction in thegrowth of cancer cells. In some embodiments, such terms refer to astabilization or reduction in the cancer stem cell population and areduction in the cancer cell population. In some embodiments, such termsrefer to a stabilization or reduction in the growth and/or formation ofa tumor. In some embodiments, such terms refer to the eradication,removal, or control of primary, regional, or metastatic cancer (e.g.,the minimization or delay of the spread of cancer). In some embodiments,such terms refer to a reduction in mortality and/or an increase insurvival rate of a patient population. In further embodiments, suchterms refer to an increase in the response rate, the durability ofresponse, or number of patients who respond or are in remission. In someembodiments, such terms refer to a decrease in hospitalization rate of apatient population and/or a decrease in hospitalization length for apatient population.

Cortisol

The estimated daily cortisol production rate in normal subjects variesbetween 4-15 mg/m² per day or, according to more recent studies between9 and 11 mg/m² per day. In order to describe the 24-hour variation inserum cortisol levels adequately, the day may be divided into, forexample, four phases. Phase 1 is a 6-hours period of minimal secretoryactivity 4 h before and 2 h after onset of sleep. Phase 2 refers to the3rd to 5th hours of sleep when there is a preliminary nocturnalsecretory episode. Phase 3 is a 4-hour main secretory phase during thelast 3 h of sleep and the first hour after wakening. Phase 4 is an11-hour phase of intermittent secretory activity when there is a slowdecline in serum levels of cortisol.

In a study by Mah et al. (Clinical Endocrinology (2004) 61, 367-375) thecircadian rhythm of serum cortisol of normal subjects is described. Peaklevels of about 400-800 mmol/1, about 150-300 mmol/1 and about 150mmol/1 are observed at about 6 am, 2 pm and 9 pm, respectively, and thelowest level is about midnight. In this study it is observed that theendogenous cortisol levels reach their highest levels within 30 minutesafter wake-up. In order to mimic the circadian rhythm, Mah et al.recommend a thrice-daily treatment regimen of hydrocortisone, the firstdose taken in the fasted state and delaying the breakfast 1-3 hours andthe other two doses taken 15-60 min before food. A trice-daily regimenis also recommended in a recent review by Czock et al. (Clin.Pharmacokinet (2005) 44, 61-98) due to the short half-life ofhydrocortisone, and for prednisolone a twice-daily regimen is preferredover a once-daily regimen.

Cortisol Test

The absence of rapid response and inexpensive testing for cortisol has,heretofore, prevented the linking of GCR antagonists (e.g., ORG 34517)to a cortisol pre-test for entry into clinical trials for GCRantagonists and will inhibit the ability to select the patients mostlikely to receive the benefit of treatment with the compounds whenavailable for clinical use. The invention provides the pairing of anaffordable, real-time cortisol test (e.g., PopTest Cortisol) which willenable the successful completion of clinical trials for this class ofdrugs as well as form the basis for their future, anticipatedtherapeutic use(s).

Conditions that may be treated using, for example, a linked salivarycortisol quantification test and GCR antagonist (e.g., ORG 34517) systeminclude, but are not limited to the following:

Major Depressive Disorder (MDD).

MDD is a psychiatric disorder which has a lifetime prevalence of around8%. One of the most consistent findings in psychiatry is that patientswith major depression present with alterations in thehypothalamic-pituitary-adrenal (HPA) axis. A significant percentage ofdepressed patients exhibit hypersecretion cortisol, as manifested byelevated plasma, cerebrospinal fluid, and salivary concentrations ofcortisol and increased urinary free cortisol. In addition, manydepressed patients exhibit a clear inability to switch off endogenouscortisol release following exogenous challenge with the potent syntheticglucocorticoid dexamethasone (the so-called dexamethasonenon-suppressors) (Gold P. W., et al., Clinical and biochemicalmanifestations of depression: relation to neurobiology of stress. NewEngland J. Med. 319, 413-420, 1988). This ‘sub-group’ of severelycompromised patients are most often the ones in whom depression becomesa life-threatening illness that warrants hospitalization.

Other abnormalities of the HPA axis found in depressed patients areincreased cortisol response to corticotrophin, a blunted corticotrophinresponse to CRH (corticotrophin releasing hormone), and adrenal andpituitary enlargement (for a review see Holsboer, F. and Barden, N.:Endocrine Reviews 1996, 17, 187-205). These observations have beeninterpreted to suggest a causal relationship between disturbedfunctioning of the HPA axis and the pathology of depression (Murphy, B.E. P. J. of Steroid Biochem. and Mol. Biol. 1991, 38, 537-559).Therapeutic efficacy of classical antidepressants has been shown to bepreceded by or to coincide with restoration of the disturbed HPA axis indepression (Holsboer and Barden, 1996, supra). It has been postulatedthat any intervention which can restore this HPA dysfunction may haveantidepressant potential.

One type of such intervention, studies of which support the impressionthat HPA-axis functioning and high circulating cortisol is a majorinstigator of major depression is the administration of glucocorticoidsynthesis inhibitors, as has been shown in patients suffering fromCushing's Syndrome, which is a condition in which high cortisol levelsare reported as a result of adrenal gland malfunction (due to apituitary tumour or a secondary tumour, both producing the cortisolsecretagogue ACTH). The depressive symptoms associated with Cushing'sdisappear relatively quickly with the return of cortisol levels tonormal. Such treatment may involve removal of the offending tumour ortreatment with cortisol synthesis inhibitors such as metyrapone,ketoconozole, or aminoglutethimide (Murphy, B. E. P., Steroids andDepression. J. Steroid Biochem & Mol. Biol. 38, 537-558, 1991).Similarly, relatively recent clinical trials have demonstrated thatcortisol synthesis inhibitors can be used to ameliorate depressivesymptoms in severe, treatment-resistant non-Cushing depressives (Murphy,B. E. P., Can. J. Psych. 43, 279-286, 1998; see also U.S. Pat. No.4,814,333 (Ravaris, C. L.)).

Another type of intervention is the use of direct GCR antagonists, whichhave much more specific pharmacological effects as compared to synthesisinhibitors and which may help restore HPA activity Small scale pilotclinical studies have been conducted in order to study theantidepressant activity of the non-selective glucocorticoid receptorantagonist RU 486 (mifepristone; Murphy, B. E. P. et al. J. Psychiat.Neurosc. 18, 209-213, 1993). More recently (Nemeroff, C., RemeronScientific Expert Meeting, Budapest, March 29-Apr. 1, 2001) it wasdemonstrated in a Phase IIB continuation of this study, that both thenumber of responders as well as the efficacy of the psychosis treatmentincreased with increasing daily dose of mifepristone as measured by thechange in Brief Psychiatric Rating Scale (50 mg-33% change; 600 mg-40%change and 1200 mg-52% change). These data indicate that a higher doseof glucocorticoid receptor antagonist is correlated with a higherclinical efficacy.

Non-response to standard treatments, however, reach levels as high as50%. (Connolly K R, Drugs. 2011; 71: 43-64.) Frequently, extrainterventions are necessary to get patients to achieve remission.Various augmentation and combination strategies have been described inthe literature for difficult to treat major MMD patients.

Use of an HPA-axis modulating drug in these patients has not beenstudied in spite of the fact that there is clear evidence that at leasta sub-group of MDD patients have significant HPA-axis dysfunction, asnoted above. Biological symptoms, indicative of excessive activity ofthe HPA-axis, have been reported with great consistency. In parallel,there is a body of evidence suggesting that there is an associationbetween HPA-axis functioning and treatment response, where high HPA-axisactivation at baseline, or post-treatment, is associated with a poorerresponse to SSRI treatment or a higher relapse risk.

Preclinical studies indicate that HPA-axis dysfunction of the type seenin affective disorders can attenuate the neurochemical effects of aselective serotonin re-uptake inhibitor (SSRI) antidepressant.Conversely, in animals with normal HPA axis function, co-administrationof GR antagonists augmented the neurochemical effects of an S SRI. Thesedata provide a mechanistic underpinning of the GR antagonistaugmentation strategy, and moreover indicate that the strategy may proveefficacious in patients both with and without HPA axis dysfunction.

Small scale pilot clinical studies were conducted in order to study theantidepressant activity of the non-selective glucocorticoid receptorantagonist RU 486 (mifepristone; Murphy, B. E. P. et al. J. Psychiat.Neurosc. 18, 209-213, 1993). A double blind, 4 week, paroxetinecontrolled study of ORG 34517 in depressed patients was carried out.Paroxetine is a selective serotonin re-uptake inhibitor which isrecognized as an effective antidepressant for major depression. Patientswere selected which had a primary depressive disorder fulfilling thediagnostic criteria of a MDD as defined by the DSM-IV for recurrent(296.3) episodes, and who had a severity of depression which resulted ina total score of at least 22 on the HAMD-21 (Hamilton Rating Scale forDepression; see Hamilton, M. “A rating scale for depression.” J. Neurol.Neurosurg. Psychiat. 1960, 23, 56-62) scale at baseline. Patients had anepisode of depression which had lasted at least 2 weeks before baseline.

In this study, patients were randomly allocated to one of threetreatment groups. Group I patients (50 patients) received 2 capsuleswith 75 mg of ORG 34517 and one placebo (total daily dose 150 mg) forthe first 2 weeks and 2 capsules with 75 mg ORG 34517 and 1 capsule with150 mg (total daily dose 300 mg) the next 2 weeks; Group II patients (46patients) received 3 capsules with 150 mg ORG 34517 (total daily dose450 mg) in the first 2 weeks and 4 capsules of ORG 34517 (total dailydose 600 mg) in the next 2 weeks; Group III patients (44 patients)received 2 capsules with 10 mg paroxetine and one placebo capsule (totaldaily dose 20 mg) for the first 2 weeks, followed by 2 capsules of 10 mgand one capsule of 20 mg paroxetine (total daily dose 40 mg) in the next2 weeks. Medication was administered orally in the morning. Efficacyassessment was done on days 4, 7, 10, 14, 21, 28 and 35 by using the21-item HAMD scale.

Thus, GCR antagonist therapy could prove a useful mechanism fortreatment of selected individuals who fail to respond to currentanti-depressant therapies such as SSRIs, providing a way to enhanceresponsiveness or as an alternate means of achieving a maintainedeuthymia.

Psychotic Depression.

Psychotic major depression has long been recognized as a distinctpsychiatric illness, having both psychotic and depressive components ina differential diagnosis. Psychotic major depression is very common. Ithas been estimated that twenty five percent of depressed patientsadmitted to the hospital have psychotic major depression (Coryell (1984)J. Nerv. Ment. Dis. 172:521). Like major depression, psychoticdepression is often also a result of high circulating cortisol levels.Various evidence supports this concept. Psychosis has been associatedwith Cushing's syndrome (Gerson (1985) Can. J. Psychiatry 30:223-224;Saad (1984) Am. J. Med. 76:759-766). A GR antagonist has been used totreat acute psychiatric disturbances secondary to Cushing's syndrome.One study showed that a relatively high dose of such a GR antagonist(400 to 800 mg per day) was useful in rapidly reversing acute psychosisin patients with severe Cushing's' Syndrome due to adrenal cancers andectopic secretion of ACTH from lung cancer (Van der Lely (1991) Ann.Intern. Med. 114:143; Van der Lely (1993) Pharmacy World & Science15:89-90; Sartor (1996) supra). Relatively high dose mifepristone, inthe range of 8-12 mg/kg/day, over a relatively short period of time (4days), was also shown to be effective in the treatment of psychosisassociated with psychotic major depression (International PatentApplication WO 99/17779; Schatzberg and Belanoff).

Surgery-Associated Immune Suppression in the Elderly.

In healthy, young to middle aged subjects suffering from stress, thereis a physiological balance between pro-inflammatory andanti-inflammatory mediators. In the elderly, the immune response isblunted as a result of the decline in several components of the immunesystem (immune senescence) and a shifting to a chronic pro-inflammatorystatus (the so-called “inflammaging” effect (Butcher and Lord, (2004)Aging Cell, pp. 151-160). As production of cortisol remains reasonablyconstant with age, whereas summed levels of DHEA and DHEAS decreasegradually from the third decade, reaching 10-20% of their maximum by theeighth decade, Butcher and Lord (2004, supra) propose a model for ageand stress, in which the age-related increase in the ratio of cortisolto DHEAS, combined with an elevated cortisol release during stress,leads to a significant reduction of immunity in aging subjects. This isproposed to explain that aging subjects are far more prone to infectionsunder conditions of stress. (Butcher and Lord (2004, supra); Butcher etal. (2005, Aging Cell 5, pp. 319-324).

The present invention relates to the use of a GCR antagonist for theprevention or treatment of infections or infectious conditions, in anaging patient, such as a human subject. The beneficial effects of saidGCR antagonists may be explained on the basis of their correctinginfluence on the cortisol/DHEA(S) ratio. It is believed that the effectin selected subjects, found to have high circulating cortisol by asaliva test as provided for by this invention, can be explained by theunbalanced immunosuppressive role of the increased cortisol/DHEAS ratioin the aged group in comparison to the balanced influence of cortisoland DHEAS on the immune system in normal subjects.

The meaning of the term ‘aging subject’ or ‘aged subject’ will be wellunderstood in the context of the use according to this invention.Although it is not linked to an exact lower age limit this generalnotion refers in the human situation usually to a person of at least 55years old, but it is more clear with a lowest age limit set at 60, 65,70 or 75 years.

In the context of the invention, the infection or infectious conditioncan be caused by any of several agents, e.g., by bacteria, by viruses orby fungi. Also in the context of the present invention, the expression“infectious conditions” means silent or subclinical infections as wellas conditions not resulting in a manifest infectious disease, but inwhich at least one parameter associated with an infectious disease, suchas the white blood (e.g., neutrophil, basophil or eosinophil) cellcounts or the level of some antibodies or some cytokines is higher thannormal. Normal values are known to the expert and may be found instandard medical manuals.

Particular uses according to the invention relate to aging subjectssuffering from an infection or an infectious condition concomitant tostress resulting from a trauma. The invention particularly relates touses wherein the subject suffers from the consequences of a bonefracture and/or bone surgery, either for such injury or for jointreplacement for osteoarthritis or rheumatoid arthritis. The inventionalso relates to uses wherein the subject suffers from an infection or aninfectious condition concomitant to psychological stress, particularlyacute emotional stress.

Post Traumatic Stress Disorder (PTSD).

PTSD is a severe anxiety disorder that can develop after exposure to anyevent that results in psychological trauma. This event may involve thethreat of death to oneself or to someone else, or to one's own orsomeone else's physical, sexual, or psychological integrity,overwhelming the individual's ability to cope. As an effect ofpsychological trauma, PTSD is less frequent and more enduring than themore commonly seen acute stress response. Diagnostic symptoms for PTSDinclude re-experiencing the original trauma(s) through flashbacks ornightmares, avoidance of stimuli associated with the trauma, andincreased arousal, such as difficulty falling or staying asleep, anger,and hyper-vigilance. Formal diagnostic criteria (both DSM-IV-TR andICD-9) require that the symptoms last more than one month and causesignificant impairment in social, occupational, or other important areasof functioning. (Diagnostic and statistical manual of mental disorders:DSM-IV. American Psychiatric Association. 1994. Washington, D.C.:American Psychiatric Association.)

PTSD displays biochemical changes in the brain and body that differ fromother psychiatric disorders such as major depression. Abundant evidencesuggests derangement of HPA-axis physiology in individuals diagnosedwith PTSD, though the nature of the derangements is variable: some havelow cortisol, some have normal levels, others have high levels ofcortisol and for some, levels may be normal, but circadian rhythm islost. It is postulated that these reflect different baseline mechanisms,but that when cortisol is high, either in a sustained way through theday or by loss of circadian rhythm with elevated night time levels, itis likely to be an important component of the clinical symptomatology(Lindley S E, et al. Basal and dexamethasone suppressed salivarycortisol concentrations in a community sample of patients withposttraumatic stress disorder. Biol. Psychiatry 2004; 55: 940-5). Insuch patients, determined by salivary cortisol testing, administrationof a GCR antagonist is expected to be therapeutic or beneficial for thesymptoms of PTSD.

Prevention of Weight Gain in Patients Using Anti-Psychotic andAnti-Depressant Medications.

Anti-psychotic and some anti-depressant medications (e.g., SSRIs) areamongst the most important tools for treating psychiatric conditions ofall kinds. However, management of patients on who take many of thesemedications for chronic, long term disease is made difficult by theirsignificant side effect profiles. One of the most important of these isweight gain and the attendant metabolic syndrome that follows. Forexample, it is estimated that 40-80% of patients who are under chronicanti-psychotic administration experience substantial weight gain, oftenexceeding 20% or more over their ideal body weights (Umbricht et al. JClin. Psychiatry 1994; 55: 157-160; Khan A Y, et al. J Psychiatr Pract.2010; 16: 289-96; Pramyothin P, Khaodhiar L. Curr Opin EndocrinolDiabetes Obes. 2010; 17: 460-6.; Rummel-Kluge C et al. Schizophr Res.2010; 123: 225-33). Such weight gain is one of the most common causes ofpoor compliance with anti-psychotic and anti-depressant regimens and,therefore, of long term failure of therapy. Furthermore, anti-psychoticmedications specifically are commonly associated with development ofinsulin resistance and metabolic syndrome (with development of type 2diabetes mellitus and hyper/dyslipidemia states) and the potentially andsignificantly increased risks for cardiovascular disease; theseconditions are of tremendous medical consequence for patients who arethereby caught in a “can't live with them, can't live without them”treatment scenario. While weight gain is potentially seen with allanti-psychotic medications, they are particularly common and tend tomore severe with the newer or “atypical” AP drugs (Allison et al. Am JPsychiatry 1999; 156:1686-1696; Rummel-Kluge C et al. Schizophr Res.2010; 123: 225-33).

Elevations in cortisol are associated with changes in body fat andinsulin resistance. Several years ago, in a proof of principle clinicalexperiment, it was reported that one GCR anatogonist (mifepristone) wasa highly effective treatment for multiple medical complications in apatient with Cushing's disease whose illness had not responded tosurgery and radiation, including reversal of insulin dependent diabetes:the patient was able to stop insulin within a month (Chu et al., J.Clin. Endocrinol. Metab. 2001; 86, 3568-3573.). These data suggest thata GCR antagonist could be useful for blocking and reversing the insulinresistance and weight changes seen in some patients treated withatypical antipsychotic agents. To this end, this compound was tested inrats who had olanzapine-induced weight gain and increases in abdominalfat; reversal of weight gain was seen and reduction of abdominal fat wasobtained (Beebe et al. Behav. Brain Res. 2006; 171, 225-229). A clinicaltrial with this compound then confirmed this benefit in humans with a 2week study of 600 mg/day of mifepristone that reduced olanzapine-inducedweight gain in 57 non-overweight healthy males with Body Mass Indicesless than 25 (Gross et al., Adv Ther. 2009; 26: 959-69.). Thus, GCRantagonist therapy could prove a useful mechanism to target in treatingpsychotic patients with atypical antipsychotic agents.

Cushing's Syndrome.

Cushing's Syndrome is a set of conditions in which high levels ofcirculating cortisol or other GCR agonists cause a set of seriouslydebilitating and sometimes life threatening signs and symptomsincluding, but not limited to, psychiatric disturbances (e.g. anxiety,depression, psychosis), immunosuppression, insulin resistance andmetabolic syndrome, skin conditions, hypertension and osteoporosis.Endogenous cortisol may be produced by ACTH-secreting, benign ormalignant tumors of the pituitary gland (“Cushing's Disease”) or of theadrenal cortex. These are rare conditions and therefore Cushing'sSyndrome is considered an “orphan disease.”

A proof of concept trial using RU486 to treat patients withtumor-related Cushing's Syndrome demonstrated efficacy in remittingsymptoms such as glucose metabolic abnormalities (i.e., glucoseintolerance; (group 1) and hypertension (group 2). Statisticallysignificant improvement was achieved for both groups: with 60%responding in the glucose intolerant group and 43% in the hypertensivegroup (Corcept Therapeutics Press Release Dec. 22, 2010). Thus, GCRantagonist therapy can be expected to provide clinical benefits forpatients with Cushing's Syndrome administered prior to tumor surgery toimprove surgical outcomes and/or post-surgery to mitigate symptoms inpatients for whom surgical cure is not achievable.

In addition, GCR antagonist therapy can be expected to provide clinicalbenefits for patients, for example, in hospitals, nursing homes,nurseries, daycares, schools, work environments, public transportation,healthcare settings, psychiatric institutions, and long-term nursingfacilities

Diagnostic Systems and Kits

A diagnostic kit may comprise some or all of the followingcomponents: 1) one or more standards comprised of one or more of thebiomarker(s) of the invention, such as cortisol; 2) a ligand, such as anantibody or a plurality of antibodies, that are specific for thebiomarker(s) that are to be assayed for using the kit; 3) writteninstructions; 4) diluents for samples and the standards; 5) a washbuffer; 6) color reagents; 7) stop solution; and 8) a ligand carrier,such as an antibody carrier, for example, a lateral flow device, or amicroplate with bound antibody, or polystyrene beads.

An example of such a kit is a quantitative ELISA (enzyme-linkedimmunosorbent assay) that determines the concentration or concentrationsof the biomarker or biomarker(s) in accordance with methods embodied bythe invention. The principle of the assay is to use the quantitativesandwich enzyme immunoassay technique wherein a monoclonal or polyclonalantibody selective for a biomarker is pre-coated onto a carrier such asa microplate into its wells. The standards and sample are then pipettedinto the wells and any of the biomarker that is present is bound to thisimmobilized antibody. Next, the wells are washed with washing buffer,and an enzyme-linked monoclonal or polyclonal antibody that is specificfor the biomarker is added to the wells. Washing is again performed,then a substrate solution is added to the wells. Color subsequentlydevelops in proportion to the amount of polypeptide of the inventionthat is bound in the first step. The color development is stopped usinga stop solution, and the intensity of the color is measured by amicroplate reader.

The methods of the invention may be carried out using, for example, alateral flow assay. Such lateral flow assays have the potential to be acost-effective, fast, simple, and sensitive method, for instance foron-site screening assays. The lateral flow assay comprises a carrierthat allows a lateral flow to occur wherein either the sample or thedetection reagent is displaced form one location on the carrier toanother. There are many formats of lateral flow assays suitable for usein a method embodied by the invention, and the skilled person willreadily know how to select and optimize a particular format. An exampleof a lateral flow test strip of the invention comprises, for example,the following components:

1. Sample pad—an absorbent pad onto which the test sample is applied.

2. Conjugate or reagent pad—this contains antibodies specific to thetarget analyte conjugated to colored particles (usually colloidal goldparticles, or latex microspheres).

3. Reaction membrane—typically a hydrophobic nitrocellulose or celluloseacetate membrane onto which anti-target analyte antibodies areimmobilized in a line across the membrane as a capture zone or test line(a control zone may also be present, containing antibodies specific forthe conjugate antibodies).

4. Wick or waste reservoir—a further absorbent pad designed to draw thesample across the reaction membrane by capillary action and collect it.

Double Antibody Sandwich Assays—

In this format the sample migrates from the sample pad through theconjugate pad where any target analyte present will bind to theconjugate. The sample then continues to migrate across the membraneuntil it reaches the capture zone where the target/conjugate complexwill bind to the immobilized antibodies producing a visible line on themembrane. The sample then migrates further along the strip until itreaches the control zone, where excess conjugate will bind and produce asecond visible line on the membrane. This control line indicates thatthe sample has migrated across the membrane as intended. Two clear lineson the membrane is a positive result. A single line in the control zoneis a negative result. Double antibody sandwich assays are most suitablefor larger analytes, such as bacterial pathogens and viruses, withmultiple antigenic sites. Competitive assays are primarily used fortesting small molecules and differ from the double antibody sandwichformat in that the conjugate pad contains antibodies that are alreadybound to the target analyte, or to an analogue of it. If the targetanalyte is present in the sample it will therefore not bind with theconjugate and will remain unlabelled. As the sample migrates along themembrane and reaches the capture zone, an excess of unlabelled analytewill bind to the immobilized antibodies and block the capture of theconjugate, so that no visible line is produced. The unbound conjugatewill then bind to the antibodies in the control zone producing a visiblecontrol line. A single control line on the membrane is a positiveresult. Two visible lines in the capture and control zones is a negativeresult. However, if an excess of unlabelled target analyte is notpresent, a weak line may be produced in the capture zone, indicating aninconclusive result. Competitive assays are most suitable for testingfor small molecules, such as mycotoxins, unable to bind to more than oneantibody simultaneously. There are a number of variations on lateralflow technology. The capture zone on the membrane may containimmobilized antigens or enzymes—depending on the target analyte—ratherthan antibodies. It is also possible to apply multiple capture zones tocreate a multiplex test. For example, commercial test strips able todetect both EHEC Shiga toxins ST1 and ST2 separately in the same samplehave been developed. Lateral flow immunoassays are simple to use byuntrained operators and generally produce a result within 15 minutes.They are very stable and robust, have a long shelf life and do notusually require refrigeration. They are also relatively inexpensive toproduce. These features make them ideal for use at the point-of-care andfor testing samples in the field, as well as in the laboratory. However,their sensitivity is limited without additional concentration or cultureprocedures.

Quantitative Tests—

While most lateral flow immunoassays are only capable of providing aqualitative result, it is possible to obtain some degree ofquantification by measuring the amount of conjugate bound to the capturezone. This can be done using a dedicated reader to measure the intensityof the colored test line. For example, the Neogen Corporation hasdeveloped the Accuscan™ lateral flow reader for use with its range ofReveal® assay kits and Charm Sciences also supplies a reader for itsRosa® range of mycotoxin test strips. More sophisticated techniques,such as fluorescent dye labeled conjugates, have also been developed toimprove the quantitative potential of lateral flow assays. Applicationsin the 20 years since the first lateral flow test was launched haveexpanded to include a huge range of different tests that have beendeveloped based on the same technology. The first commercially availablekits were aimed at the clinical diagnostics field, but there are nowproducts with applications in almost every branch of microbiology.Clinical microbiology—lateral flow tests have been developed forbacterial pathogens, respiratory and enteric viruses, intestinalparasites and bacterial toxins. Many of the lateral flow immunoassayproducts designed for the clinical sector were intended to be used atthe point-of-care for direct testing of fecal, blood and urine samplesand nose and throat swabs, where the simple operation and speed of thetests is key to their use outside of the laboratory. However, the sametest strips may also be useful as a quick confirmatory test followinglaboratory culture of clinical samples. Food and agriculturalmicrobiology—test strips are available for food borne bacterialpathogens, bacterial and fungal toxins. In the food microbiology sector,the main applications are more likely to be in the laboratory, althoughthere are field test kits for mycotoxins in grain samples. Testing forfood borne bacterial pathogens generally involves at least oneenrichment stage before the assay strip is used to confirm the presenceor absence of the pathogen. Some manufacturers, such as Dupont®, havedeveloped enrichment media and methods specifically designed for usewith lateral flow test strips. Test strips may also be useful for rapidconfirmation of the identity of bacterial isolates from conventionalmicrobiological testing.

A diagnostic system in kit form of the present invention includes, forexample, in an amount sufficient for at least one assay, a polypeptide,antibody composition or monoclonal antibody composition of the presentinvention, as a packaged reagent. Instructions for use of the packagedreagent are also typically included.

A diagnostic system in kit form of the present invention may include,for example, a means for detecting the presence of a biologicalsubstance in a test sample, comprising for example, a lollipop-likeapparatus including a stem integrated with the base and a headintegrated with the stem, for collecting a test sample consisting of,for example, saliva, or a bodily fluid sample from a subject. The stemhead may include a receptor of a sponge like carrier to ensure a highvoid volume to absorb sufficient saliva, or bodily fluid sample. SeeU.S. Pat. No. 7,993,283, incorporated by reference herein in itsentirety.

A diagnostic system in kit form of the present invention may include,for example, a means for combining the test sample with a bufferingsystem (Reagent 1) containing viscosity controllers and stabilizers intoa reaction vessel and mixing the solution. A diagnostic system in kitform of the present invention may include, for example, a means forreading the a parameter of the reaction vessel with sample and buffer,and further means for combining the test sample and buffer mixture witha fluorescence-labeled ligand (Reagent 2) to said biological substancein the reaction vessel, mixing the solution to produce an assaysolution. Furthermore, Reagent 2 may be delivered to the reaction vesselwithout further dilution volume of the assay solution.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic, paper, foil and the like capable of holdingwithin fixed limits a polypeptide, antibody composition or monoclonalantibody composition of the present invention. Thus, for example, apackage can be a glass vial used to contain milligram quantities of acontemplated polypeptide or it can be a microtiter plate well to whichmicrogram quantities of a contemplated polypeptide have been operativelyaffixed, i.e., linked so as to be capable of being immunologically boundby an antibody.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In preferred embodiments, a diagnostic system of the present inventionfurther includes a label or indicating means capable of signaling theformation of a complex containing a polypeptide or antibody molecule ofthe present invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent oneclass of gamma ray emission-producing radioactive element indicatinggroups. Particularly preferred is¹²⁵I. Another group of useful labelingmeans are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N which themselvesemit positrons. The positrons so emitted produce gamma rays uponencounters with electrons present in the animal's body. Also useful is abeta emitter, such ¹¹¹indium or ³H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795, which are all incorporated herein by reference.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect, for example, the presence or quantity of cortisol in abody fluid sample such as serum, plasma, or urine, etc. “ELISA” refersto an enzyme-linked immunosorbent assay that employs an antibody orantigen bound to a solid phase and an enzyme-antigen or enzyme-antibodyconjugate to detect and quantify the amount of an antigen or antibodypresent in a sample. A description of the ELISA technique is found inChapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P.Sites et al., published by Lange Medical Publications of Los Altos,Calif. in 1982 and in U.S. Pat. No. 3,654,090; U.S. Pat. No. 3,850,752;and U.S. Pat. No. 4,016,043, which are all incorporated herein byreference.

Thus, for example, a polypeptide, antibody molecule composition ormonoclonal antibody molecule composition of the present invention can beaffixed to a solid matrix to form a solid support that comprises apackage in the subject diagnostic systems. The reagent is typicallyaffixed to the solid matrix by adsorption from an aqueous mediumalthough other modes of affixation, well known to those skilled in theart, can be used.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include cross-linked dextran; agarose; beads ofpolystyrene beads about 1 micron to about 5 millimeters in diameter;polyvinyl chloride, polystyrene, cross-linked polyacrylamide,nitrocellulose- or nylon-based webs such as sheets, strips or paddles;or tubes, plates or the wells of a microtiter plate such as those madefrom polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems. Suchmaterials include glass and plastic (e.g., polyethylene, polypropyleneand polycarbonate) bottles, vials, plastic and plastic-foil laminatedenvelopes and the like. In one embodiment a diagnostic system of thepresent invention is useful for assaying for the presence of, forexample, cortisol. Such a system comprises, in kit form, a packagecontaining an antibody to, for example, cortisol.

“Sample” refers to, for example, essentially any source from whichmaterials of interest to be analyzed (e.g., ligands and antiligands,such as antibodies and antigens, and nucleic acids and theircomplements) can be obtained. A sample may be acquired from essentiallyany organism, including animals and plants, as well as cell cultures,recombinant cells and cell components. Samples can be from a biologicaltissue, fluid or specimen and may be obtained from a diseased or healthyorganism. Samples may include, but are not limited to, saliva, sputum,amniotic fluid, blood, blood cells (e.g., white cells), urine, semen,peritoneal fluid, pleural fluid, tissue or fine needle biopsy samples,and tissue homogenates. Samples may also include sections of tissuessuch as frozen sections taken for histological purposes. Typically,samples are taken from a human. However, samples can be obtained fromother mammals also, including by way of example and not limitation,dogs, cats, sheep, cattle, and pigs. The sample may be pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, preferably at physiological pH can be used.

Biological samples can be derived from patients using well knowntechniques such as venipuncture, lumbar puncture, fluid sample such assaliva or urine, or tissue biopsy and the like. When the biologicalmaterial is derived from non-humans, such as commercially relevantlivestock, blood and tissue samples are conveniently obtained fromlivestock processing plants. Alternatively, a biological sample may beobtained from a cell or blood bank where tissue and/or blood are stored,or from an in vitro source, such as a culture of cells. Techniques forestablishing a culture of cells for use as a source for biologicalmaterials are well known to those of skill in the art.

In one embodiment the sample is selected from or is derived from, forexample, microbial products or biological products.

Although the above described example relates to the antigens relating todisease, the immunoassay apparatus could be used, for example, as anallergy test kit, as a test kit for drugs of abuse or for analyzingnon-human derived samples e.g. bovine, porcine, and veterinary tests.

Specific reagents used in the assay device will be selected so as toensure that the particular target analyte is detected as is well knownin the art. The target analyte may be any analyte for example a chemicalreagent which may be organic or inorganic, and which optionallycomprises a hapten, a protein, a polypeptide, a microorganism or anucleic acid sequence.

In particular, the analyte is a hormone such as a fertility hormone likeprogesterone or a stress hormone such as cortisol. However, there is awide range of applications of these types of tests across the entirefield of diagnostics and analysis. Detection of marker proteins orhormones can be diagnostic of certain disease conditions in humans oranimals, and the presence of drugs or drug residues may also be requiredto be detected, for example, in animal husbandry, forensic medicine orin the testing for banned or prohibited drug substances.

Alternatively, the analyte is a chemical reagent, for instance a smallmolecule, which suitably comprises a hapten. Small molecules willgenerally comprise a single recognizable binding site. Typically theywill have a molecular weight of less than 1 kDa.

Where the assay utilizes a labelled binding partner for the analyte andthe analyte is a chemical reagent, the binding partner may comprise anyother reagent which reacts with or otherwise becomes associated with thechemical reagent, either because it forms covalent or ionic bonds withthe reagent, or by the formation of other interactions, such as hydrogenbonding or Van der Waals interactions. For example, where the chemicalreagent is an acid, the binding partner may comprise an alcohol or anamine that forms an ester or amide with the acid under the sorts ofconditions found in the test. Alternatively the binding partner maycomprise a base that forms a salt with the acid. Conversely, where thebinding partner may comprise the acid part of the reactive pair.

Where the analyte is or comprises a hapten or a protein antigen, thebinding partner may comprise an antibody or a binding fragment thereof,which may be monoclonal, polyclonal or recombinant, but preferably ismonoclonal. Where the analyte is a hormone or enzyme, the labelledbinding partner may comprise a labelled receptor for the analyte.However, where the analyte is itself an immunoglobulin, and inparticular, an antibody, the labelled binding partner may also comprisefor instance, an antigen or recombinant antigen, as well asanti-antibody immunoglobulin such as anti-sera.

Antibodies or binding fragments to small molecules such as haptens, aregenerated by attaching the molecule to an immunogenic reagent andadministering this to an animal such as a mouse or rabbit. Antibodiesare then harvested from the animal in the usual way. Monoclonalantibodies are obtained by fusing spleen cells to hybridoma cells, andselecting those which bind the hapten, using routine procedures.

For example, where the analyte is a biologically active material such asan active agrochemical as discussed above, specific reagents used in theassay device will be selected so as to ensure that the particular targetbiologically active material is detected as is well known in the art.The biologically active material may be any active chemical such as anagrochemical, for example a chemical reagent which may be organic orinorganic, and which optionally comprises a hapten, a protein, apolypeptide, a microorganism or a nucleic acid sequence. Most preferablythe biologically active material is a chemical reagent, for instance asmall molecule, which suitably comprises a hapten. Small molecules willgenerally have a single antibody binding site. Typically they will havea molecular weight of less that 1 kDa.

Antibodies or binding fragments to small molecules such as haptens, aregenerated by attaching the molecule to an immunogenic reagent andadministering this to an animal such as a mouse or rabbit. Antibodiesare then harvested from the animal in the usual way. Monoclonalantibodies are obtained by fusing spleen cells to hybridoma cells, andselecting those which bind the hapten, using routine procedures.

Microarrays

The method of the invention is particularly useful in combination withthe analysis of gene expression profiles. In some embodiments, a geneexpression profile, such as a collection of transcription rates of anumber of genes, is converted to a projected gene expression profile.The projected gene expression profile is a collection of expressionvalues. The conversion is achieved, in some embodiments, by averagingthe transcription rate of the genes. In some other embodiments, otherlinear projection processes may be used.

Microarrays may be prepared and analyzed using methods known in the art.Oligonucleotides may be used as either probes or targets in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously and to identify geneticvariants, mutations, and single nucleotide polymorphisms. Suchinformation may be used to determine gene function; to understand thegenetic basis of a condition, disease, or disorder; to diagnose acondition, disease, or disorder; and to develop and monitor theactivities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S.Pat. No. 5,474,796; Schena et al. (1996) Proc. Natd. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon et al. (1995) PCT application WO95/35505; Heller etal. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller et al.(1997); U.S. Pat. No. 5,605,662.) Hybridization probes are also usefulin mapping the naturally occurring genomic sequence. The sequences maybe mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome DNA libraries.

Using the methods of the invention a skilled artisan can readily selectand prepare probes for a microarray wherein the microarray containsspecific individual probes for less than all the genes in the genome andless than all the genes in the genome. In such embodiments, themicroarray contains one or two or more individual probes, each of whichhybridizes to an expression product (e.g., mRNA, or cDNA or cRNA derivedtherefrom) for a desired number of genes. Thus, for example, changes inthe expression of all or most of the genes in the entire genome of acell or organism can thereby be monitored by use of a surrogate and on asingle microarray by measuring expression of the group of genes that arerepresentative of all or most of the genes of the genome. Suchmicroarrays can be prepared using the selected probes and are thereforepart of the present invention.

Glucocorticoid Receptor

The glucocorticoid receptor is widely distributed and expressed in manycultured cell lines, and the control of gene expression byglucocorticoids, therefore, has been widely studied as a model fortranscriptional regulation. A number of glucocorticoid-responsivetranscription units, including mouse mammary tumor virus (MMTV)(Ringold, et al., 1975; Parks, et al., 1974), mouse and humanmetallothionein (Eager, et al., 1981; Karin, et al., 1980), ratalpha₂M-globulin (Kurtz, et al., 1977) and rat and human growth hormone(Spindler, et al., 1982; Evans, et al., 1982; Robins, et al., 1982)genes have been identified. DNA sequences mediating transcriptionalstimulation of several of these genes have been localized. For MMTV,these sequences are discrete genomic regions upstream of thetranscriptional start site which appear to exert their actionsindependently of orientation and position (Chandler, et al., 1983;Ostrowski, et al., 1984). The steroid/receptor complex appears to bindto these regulatory sequences and purified receptor has been used todefine the specific binding sites (Govinda, et al., 1982; Scheidereit,et al., 1983; Pfahl, 1982; Payvar, et al., 1983). The ability of theglucocorticoid-responsive element (GRE) to alter its position andorientation yet still maintain promoter inducibility suggests that itresembles the class of cis-acting regulatory sequences termed enhancers(Chandler, et al., 1983). First discovered in viruses and subsequentlyin cellular genes, these sequences are necessary for efficienttranscription in vivo (Laimonis, et al., 1982; Benoist, et al., 1981;Baerji, et al., 1983). It has been suggested that enhancers arerecognized by trans-acting factors that mediate regulatory effects bytissue-specific transcriptional control. Although the enhancer factorshave not been well characterized, the glucocorticoid receptor may serveas a paradigm for these putative gene activator proteins.

It is generally accepted that the unliganded glucocorticoid receptor(GR) resides in the cytoplasm, and that hormone activation leads both tonuclear accumulation and gene activation. (Gasc, J. -M. & Baulieu, E. E.(1987) in Steroid Hormone Receptors: Their Intracellular Localisation,ed. Clark, C. R. (Ellis Horwood Ltd., Chichester, England), pp. 233-250;Beato, M. (1989) Cell 56, 335-344; Carson-Jurica, M. A., Schrader, W. T.& O'Malley, B. W. (1990) Endocr. Rev. 11, 201-220; Gronemeyer, H. (1993)in Steroid Hormone Action, ed. Parker, M. G. (Oxford University Press,New York), pp. 94-117; Tsai, M. J. & O'Malley, B. W. (1994) Annu. Rev.Biochem. 63, 451-486; Akner, G., Wikstrom, A. C. & Gustafsson, J. A.(1995) J. Steroid Biochem. Mol. Biol. 52, 1-16), and references therein.However, the mechanisms involved in nuclear translocation and targetingof steroid receptors to regulatory sites in chromatin have been poorlyunderstood. It has previously been difficult to discriminate between theability of a given receptor mutant, or a given receptor/ligandcombination, to participate in the separate processes of receptoractivation, nuclear translocation, sequence-specific binding, andpromoter activation.

The glucocorticoid receptor (GR) is expressed in a subset of bothERalpha-positive and -negative human breast cancers as well as in someovarian cancers. In vitro and in vivo experiments suggest thatactivation of the GR in ER-negative pre-malignant breast epithelial andcancer cells initiates cell survival pathways under stress conditionsthat normally induce significant cell death (e.g. chemotherapy,radiation, growth factor deprivation).

The glucocorticoid receptor (GR) is present in glucocorticoid responsivecells where it resides in the cytosol in an inactive state until it isstimulated by an agonist. Upon stimulation the glucocorticoid receptortranslocates to the cell nucleus where it specifically interacts withDNA and/or protein(s) and regulates transcription in a glucocorticoidresponsive manner Two examples of proteins that interact with theglucocorticoid receptor are the transcription factors, API and NFκ-B.Such interactions result in inhibition of API- and NFκ-B-mediatedtranscription and are believed to be responsible for some of theanti-inflammatory activity of endogenously administered glucocorticoids.In addition, glucocorticoids may also exert physiologic effectsindependent of nuclear transcription. Biologically relevantglucocorticoid receptor agonists include cortisol and corticosterone.Many synthetic glucocorticoid receptor agonists exist includingdexamethasone, prednisone and prednisolone.

Glucocorticoid Receptor Antagonists

Glucocorticoid receptor antagonists bind to the receptor and preventglucocorticoid receptor agonists from binding and eliciting GR mediatedevents, including transcription. RU486 is an example of a non-selectiveglucocorticoid receptor antagonist.

Compounds having high glucocorticoid receptor binding affinity and, inaddition, high in vivo anti-glucocorticoid activity, while having, forexample, low androgenic and progestagenic activities are disclosed inU.S. Pat. No. 6,011,025, incorporated herein by reference in itsentirety. ORG 34517 is an example of a compound with high glucocorticoidreceptor binding affinity while having low androgenic and progestagenicactivities.

It has been found that 11-(substituted phenyl)-estra-4,9-dienederivatives of formula I

whereinA is a residue of a 5- or 6-membered ring containing 2 heteroatoms whichare not connected to each other and independently selected from O and S,the ring being optionally substituted with one or more halogen atoms, orA is a residue of a 5- or 6-membered ring wherein no double C—C bondsare present, containing 1 heteroatom selected from O and S, whichheteroatom is connected to the phenyl group at the position indicatedwith an asterisk, the ring being optionally substituted with one or morehalogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H, (1-8C)alkyl,halogen or CF3; X is selected from (H₂OH), O, and NOH; and theinterrupted line represents an optional bond, show specific and highglucocorticoid receptor binding affinity and are highly active in vivoshowing predominant anti-glucocorticoid activity.

The compounds lack appreciable affinity for mineralocorticoid,progesterone, estrogen and androgen receptors, indicating a clean sideeffect profile.

The 11-(substituted phenyl)-estra-4,9-diene derivatives of the inventioncan be used in the prevention and treatment of glucocorticoid dependentdiseases or symptoms, like Cushing syndrome, diabetes, glaucoma, sleepdisturbances, depression, anxiety, atherosclerosis, hypertension,adiposity, osteoporosis and withdrawal symptoms from narcotics and theirmixtures.

Preferred compounds according to this invention are 11-(substitutedphenyl) estra-4,9-diene derivatives, wherein the heteroatom(s) are (is)O, the 5- or 6-membered ring being optionally substituted with one ormore fluorine atoms; R1 is H; and X is O or NOH. More preferredcompounds are 11-(substituted phenyl) estra-4,9-diene derivativeswherein A is a residue of a 5-membered ring. Particularly preferred are11-(substituted phenyl) estra-4,9-diene derivatives wherein A contains 2heteroatoms being O.

Especially preferred are 11-(substituted phenyl) estra-4,9-dienederivatives wherein R2 is methyl and the interrupted line represents abond.

The most preferred compound is(11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (ORG 34517).

The term halogen means a fluorine, chlorine, bromine or iodine atom.Fluorine is the preferred halogen in ring A and when R2 is halogen,chlorine is preferred. The terms (1-4C)alkyl and (1-8C)alkyl, as used inthe definitions of R1 and R2, respectively, mean alkyl groups having 1-4and 1-8 carbon atoms, respectively, for example methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl,octyl.

The 11-(substituted phenyl)-estra-4,9-diene derivatives according to thepresent invention can be prepared by a process wherein a compound offormula II

wherein A, R2 and the interrupted line have the meanings as previouslydefined, R1 is H, and P is a protected keto-group, is dehydrated anddeprotected, after which the 17β-OH is optionally esterified by reactionwith an appropriate carboxylic acid to give a derivative wherein R1 is1-oxo(1-4C)alkyl, and optionally the 3-oxo group is converted into thecorresponding 3-hydroxy- or 3-oxime derivative. The 3-oxo group can bereduced to form the 3-hydroxy-derivative by using a suitable reducingagent, such as sodium borohydride. The 3-oxime derivatives can beprepared by hydroxylamine treatment in a suitable solvent, likepyridine.

The derivatives of formula II may be prepared according to well knownmethods described and used for the preparation of steroids.

A suitable process for the preparation of derivatives of formula IIstarts from estra-4,9-diene-3,17-dione. Selective reduction of the17-keto group to 17β-OH, 17α-H, e.g. with sodium borohydride, followedby protection of the 3-keto group, e.g., by ketalisation withethyleneglycol, triethylorthoformate and p-toluenesulfonic acid, andoxidation of the 17-hydroxy group, e.g. with pyridinium chlorochromate,provides the 3-ketoprotected estra-5(10),9(11)-diene-3,17-dione.Alkynylation at the 17-position (yielding a 17α-alkynyl,17β-OHderivative), followed by epoxidation of the 5(10) double bond, e.g. withhydrogen peroxide, trifluoroacetophenone, and pyridine indichloromethane according to the method as disclosed in European patentapplication EP 0 298 020, provides the 3-ketoprotected5α,10α-epoxy-17α-alkynyl-17β-hydroxy-estr-9(11)-ene-3-one.

Subsequently, compounds of formula II are formed from this epoxidederivative, for example by reaction with an organometallic compound ofthe formula

wherein X is a (alkali)metal, like lithium, or a magnesiumhalide,preferably magnesium bromide.

Suitable protective groups and methods to remove these groups are knownin the art, for example from T. W. Green: Protective Groups in OrganicSynthesis (Wiley, N Y, 1981). Particularly suitable protective groupsfor the protection of keto groups are acetals, e.g. 1,2-ethylene ketal.

The specificity of ORG 34517 for GR blockade, without significantcross-binding to other related steroidal hormone receptors (such asthose for estrogen and progesterone), eliminates the likelihood ofsignificant toxicities and side effects. Indeed, none were identified inall the substantial phase I and phase II clinical trials that alreadyhave been performed with the compound. Because the drug is envisioned asbeing used in limited dosing over time, coordinated with theintermittent dosing strategies typical for chemotherapeutic agents, theGR blockade also would not lead to significant alteration of HPA-axisfunctioning, with rapid restitution of the HPA-axis to baselinefollowing dosing.

Formulations

The compounds of the invention may be administered enterally orparenterally. Mixed with pharmaceutically suitable auxiliaries, e.g., asdescribed in the standard reference, Gennaro et al., Remington'sPharmaceutical Sciences. The compounds may be compressed into soliddosage units, such as pills, tablets, or be processed into capsules orsuppositories. By means of pharmaceutically suitable liquids thecompounds can also be applied in the form of a solution, suspension,emulsion, e.g. for use as an injection preparation or eye drops, or as aspray, e.g. for use as a nasal spray.

For making dosage units, e.g., tablets, the use of conventionaladditives such as fillers, colorants, polymeric binders and the like iscontemplated. In general, any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

Dosage Forms

The compositions of the present invention can be processed byagglomeration, air suspension chilling, air suspension drying, balling,coacervation, coating, comminution, compression, cryopelletization,encapsulation, extrusion, wet granulation, dry granulation,homogenization, inclusion complexation, lyophilization, melting,microencapsulation, mixing, molding, pan coating, solvent dehydration,sonication, spheronization, spray chilling, spray congealing, spraydrying, or other processes known in the art. The compositions can beprovided in the form of a minicapsule, a capsule, a tablet, an implant,a troche, a lozenge (minitablet), a temporary or permanent suspension,an ovule, a suppository, a wafer, a chewable tablet, a quick or fastdissolving tablet, an effervescent tablet, a buccal or sublingual solid,a granule, a film, a sprinkle, a pellet, a bead, a pill, a powder, atriturate, a platelet, a strip or a sachet. Compositions can also beadministered as a “dry syrup”, where the finished dosage form is placeddirectly on the tongue and swallowed or followed with a drink orbeverage. These forms are well known in the art and are packagedappropriately. The compositions can be formulated for oral, nasal,buccal, ocular, urethral, transmucosal, vaginal, topical or rectaldelivery.

The pharmaceutical composition can be coated with one or more entericcoatings, seal coatings, film coatings, barrier coatings, compresscoatings, fast disintegrating coatings, or enzyme degradable coatings.Multiple coatings can be applied for desired performance. Further, thedosage form can be designed for immediate release, pulsatile release,controlled release, extended release, delayed release, targeted release,synchronized release, or targeted delayed release. Forrelease/absorption control, solid carriers can be made of variouscomponent types and levels or thicknesses of coats, with or without anactive ingredient. Such diverse solid carriers can be blended in adosage form to achieve a desired performance. The definitions of theseterms are known to those skilled in the art. In addition, the dosageform release profile can be affected by a polymeric matrix composition,a coated matrix composition, a multiparticulate composition, a coatedmultiparticulate composition, an ion-exchange resin-based composition,an osmosis-based composition, or a biodegradable polymeric composition.Without wishing to be bound by theory, it is believed that the releasemay be effected through favorable diffusion, dissolution, erosion,ion-exchange, osmosis or combinations thereof.

When formulated as a capsule, the capsule can be a hard or soft gelatincapsule, a starch capsule, or a cellulosic capsule. Although not limitedto capsules, such dosage forms can further be coated with, for example,a seal coating, an enteric coating, an extended release coating, or atargeted delayed release coating. These various coatings are known inthe art, but for clarity, the following brief descriptions are provided:seal coating, or coating with isolation layers: Thin layers of up to 20microns in thickness can be applied for variety of reasons, includingfor particle porosity reduction, to reduce dust, for chemicalprotection, to mask taste, to reduce odor, to minimize gastrointestinalirritation, etc. The isolating effect is proportional to the thicknessof the coating. Water soluble cellulose ethers are preferred for thisapplication. HPMC and ethyl cellulose in combination, or Eudragit E100,may be particularly suitable for taste masking applications. Traditionalenteric coating materials listed elsewhere can also be applied to forman isolating layer.

Extended release coatings are designed to effect delivery over anextended period of time. The extended release coating is apH-independent coating formed of, for example, ethyl cellulose,hydroxypropyl cellulose, methylcellulose, hydroxymethyl cellulose,hydroxyethyl cellulose, acrylic esters, or sodium carboxymethylcellulose. Various extended release dosage forms can be readily designedby one skilled in art to achieve delivery to both the small and largeintestines, to only the small intestine, or to only the large intestine,depending upon the choice of coating materials and/or coating thickness.

Enteric coatings are mixtures of pharmaceutically acceptable excipientswhich are applied to, combined with, mixed with or otherwise added tothe carrier or composition. The coating may be applied to a compressedor molded or extruded tablet, a gelatin capsule, and/or pellets, beads,granules or particles of the carrier or composition. The coating may beapplied through an aqueous dispersion or after dissolving in appropriatesolvent. Additional additives and their levels, and selection of aprimary coating material or materials will depend on the followingproperties: 1. resistance to dissolution and disintegration in thestomach; 2. impermeability to gastric fluids and drug/carrier/enzymewhile in the stomach; 3. ability to dissolve or disintegrate rapidly atthe target intestine site; 4. physical and chemical stability duringstorage; 5. non-toxicity; 6. easy application as a coating (substratefriendly); and 7. economical practicality.

Dosage forms of the compositions of the present invention can also beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the lowergastrointestinal tract. The enteric coated dosage form may be acompressed or molded or extruded tablet/mold (coated or uncoated)containing granules, pellets, beads or particles of the activeingredient and/or other composition components, which are themselvescoated or uncoated. The enteric coated oral dosage form may also be acapsule (coated or uncoated) containing pellets, beads or granules ofthe solid carrier or the composition, which are themselves coated oruncoated.

Delayed release generally refers to the delivery so that the release canbe accomplished at some generally predictable location in the lowerintestinal tract more distal to that which would have been accomplishedif there had been no delayed release alterations. The preferred methodfor delay of release is coating. Any coatings should be applied to asufficient thickness such that the entire coating does not dissolve inthe gastrointestinal fluids at pH below about 5, but does dissolve at pHabout 5 and above. It is expected that any anionic polymer exhibiting apH-dependent solubility profile can be used as an enteric coating in thepractice of the present invention to achieve delivery to the lowergastrointestinal tract. Polymers for use in the present invention areanionic carboxylic polymers.

Shellac, also called purified lac, a refined product obtained from the,resinous secretion of an insect. This coating dissolves in media ofpH>7.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants,stabilizers such as hydroxy propyl cellulose, acid/base may be added tothe coatings besides plasticizers to solubilize or disperse the coatingmaterial, and to improve coating performance and the coated product.

In carrying out the method of the present invention, the combination ofthe invention may be administered to mammalian species, such as dogs,cats, humans, etc. and as such may be incorporated in a conventionalsystemic dosage form, such as a tablet, capsule, elixir or injectable.The above dosage forms will also include the necessary carrier material,excipient, lubricant, buffer, antibacterial, bulking agent (such asmannitol), anti-oxidants (ascorbic acid of sodium bisulfite) or thelike.

The dose administered must be carefully adjusted according to age,weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result.

The pharmaceutical compositions of the invention may be administered inthe dosage forms in single or divided doses of one to four times daily.It may be advisable to start a patient on a low dose combination andwork up gradually to a high dose combination.

Tablets of various sizes can be prepared, e.g., of about 1 to 2000 mg intotal weight, containing one or both of the active pharmaceuticalingredients, with the remainder being a physiologically acceptablecarrier of other materials according to accepted pharmaceuticalpractice. These tablets can be scored to provide for fractional doses.Gelatin capsules can be similarly formulated.

Liquid formulations can also be prepared by dissolving or suspending oneor the combination of active substances in a conventional liquid vehicleacceptable for pharmaceutical administration so as to provide thedesired dosage in one to four teaspoonful.

Dosage forms can be administered to the patient on a regimen of, forexample, one, two, three, four, five, six, or other doses per day

In order to more finely regulate the dosage schedule, the activesubstances may be administered separately in individual dosage units atthe same time or carefully coordinated times. Since blood levels arebuilt up and maintained by a regulated schedule of administration, thesame result is achieved by the simultaneous presence of the twosubstances. The respective substances can be individually formulated inseparate unit dosage forms in a manner similar to that described above.

In formulating the compositions, the active substances, in the amountsdescribed above, may be compounded according to accepted pharmaceuticalpractice with a physiologically acceptable vehicle, carrier, excipient,binder, preservative, stabilizer, flavor, etc., in the particular typeof unit dosage form.

Illustrative of the adjuvants which may be incorporated in tablets arethe following: a binder such as gum tragacanth, acacia, corn starch orgelatin; an excipient such as dicalcium phosphate or cellulose; adisintegrating agent such as corn starch, potato starch, alginic acid orthe like; a lubricant such as stearic acid or magnesium stearate; asweetening agent such as sucrose, aspartame, lactose or saccharin; aflavoring agent such as orange, peppermint, oil of wintergreen orcherry. When the dosage unit form is a capsule, it may contain inaddition to materials of the above type a liquid carrier such as a fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets orcapsules may be coated with shellac, sugar or both. A syrup of elixirmay contain the active compound, water, alcohol or the like as thecarrier, glycerol as solubilizer, sucrose as sweetening agent, methyland propyl parabens as preservatives, a dye and a flavoring such ascherry or orange.

One embodiment of this invention includes methods of treating,preventing, or diagnosing a particular disease or condition byadministering the disclosed nanoparticles, composite nanoparticles,nanosuspension, or nanocapsules to a subject. In many instances, thenanoparticles, composite nanoparticles, or nanocapsules are administeredalone or can be included within a pharmaceutical composition. Aneffective amount of a pharmaceutical composition, generally, is definedas that amount sufficient to ameliorate, reduce, minimize, or limit theextent of the disease or condition. More rigorous definitions may apply,including elimination, eradication, or cure of the disease or condition.

“Nanoparticles” are solid particles of an average particle diameter of,for example, less than about 1 micron (micrometer). One micron is 1,000nanometers (nm).

“Stabilized” nanoparticles are nanoparticles coated with a stabilizingmaterial and having a reduced tendency for aggregation and loss ofdispersion with respect to nanoparticles of the compound of theinvention without a stabilizing coating.

A nano-spray is a spray containing nanoparticles or a spray thatproduces nanoparticles. A nanodispersion is a dispersion containingnanoparticles. A nanosuspension is a suspension containingnanoparticles.

The liquid formulations useful herein may comprise a solvent, solution,suspension, microsuspension, nanosuspension, emulsion, microemulsion,gel or even a melt containing the active component or components. Insome embodiments the nanoparticles, nanofibers, or nanofibrils may be inthe form of, or within or on, granules, powders, suspensions, solutions,dissolvable films, mats, webs, tablets, or releasable forms particularlyreleasable dosage forms. Other particular useful forms are concentratesto which a diluting liquid is added prior to use. The product may alsobe sprayed onto the inner surface of a container to which a liquid isadded later prior to use and the nanoparticles, nanofibers, ornanofibrils, are released into the liquid.

Pharmaceutical compositions of the present invention can includenanoparticles, composite nanoparticles, nanosuspension, or nanocapsulesof the present invention.

In certain non-limiting embodiments, pharmaceutical compositions maycomprise, for example, at least about 0.1% of an active ingredient ornanoparticles, composite nanoparticles, or nanocapsules, for example. Inother embodiments, the an active ingredient or nanoparticles, compositenanoparticles, or nanocapsules may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In non-limiting examples of aderivable range from the numbers listed herein, a range of about 5mg/kg/body weight to about 100 mg/kg/body weight, about 5microgram/kg/body weight to about 500 milligram/kg/body weight, etc.,can be administered.

The composition may also include various antioxidants to retardoxidation of one or more active ingredient or nanoparticles, compositenanoparticles, nanosuspension, or nanocapsules. The prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

In order to increase the effectiveness of a treatment with thenanoparticles, nanogels, composite nanoparticles, nanosuspension, ornanocapsules of the present invention, it may be desirable to combinethese nanoparticles, composite nanoparticles, or nanocapsules with othertherapies effective in the treatment of a particular disease orcondition.

The formulations as described above may be administered for a prolongedperiod, that is, for as long as the potential for a disease or conditionremains or the symptoms continue.

Packaging/Treatment Kits

The present invention relates to a kit for conveniently and effectivelycarrying out the methods in accordance with the present invention. Suchkits may be suited for the delivery of solid oral forms such as tabletsor capsules. Such a kit may include a number of unit dosages. Such kitscan include a means for containing the dosages oriented in the order oftheir intended use. An example of a means for containing the dosages inthe order of their intended uses is a card. An example of such a kit isa “blister pack”. Blister packs are well known in the packaging industryand are widely used for packaging pharmaceutical unit dosage forms. Ifdesired, the blister can be in the form of a childproof blister, i.e., ablister that is difficult for a child to open, yet can be readily openedby an adult. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarfeature and/or calendar insert, designating the days and the sections ofa day in the treatment schedule in which the dosages can beadministered, such as an AM dose is packaged with a “mid day” and a PMdose; or an AM dose is packaged with a PM dose. Alternatively, placebodosages, or vitamin or dietary supplements, either in a form similar toor distinct from the pharmaceutical active dosages, can be included.

In one aspect, the package, kit or container comprises a “blisterpackage” (also called a blister pack, or bubble pack). In one aspect,the blister package consists two or more separate compartments: Amdosage of this invention, and PM dosage of this invention, or mid-daydosage of this invention. This blister package is made up of twoseparate material elements: a transparent plastic cavity shaped to theproduct and its blister board backing. These two elements are thenjoined together with a heat sealing process which allows the product tobe hung or displayed. Exemplary types of “blister packages” include:Face seal blister packages, gang run blister packages, mock blisterpackages, interactive blister packages, slide blister packages.

Blister packs, clamshells or trays are forms of packaging used forgoods; thus, the invention provides for blister packs, clamshells ortrays comprising a composition (e.g., a (the multi-ingredientcombination of drugs of the invention) combination of activeingredients) of the invention. Blister packs, clamshells or trays can bedesigned to be non-reclosable, so consumers can tell if a package hasalready opened. They are used to package for sale goods where producttampering is a consideration, such as the pharmaceuticals of theinvention. In one aspect, a blister pack of the invention comprises amoulded PVC base, with raised areas (the “blisters”) to contain thetablets, pills, etc. comprising the combinations of the invention,covered by a foil laminate. Tablets, pills, etc. are removed from thepack either by peeling the foil back or by pushing the blister to forcethe tablet to break the foil. In one aspect, a specialized form of ablister pack is a strip pack.

In one aspect, a blister pack also comprises a method of packaging wherethe compositions comprising combinations of ingredients of the inventionare contained in-between a card and a clear PVC. The PVC can betransparent so the item (pill, tablet, geltab, etc.) can be seen andexamined easily; and in one aspect, can be vacuum-formed around a mouldso it can contain the item snugly and have room to be opened uponpurchase. In one aspect, the card is brightly colored and designeddepending on the item (pill, tablet, geltab, etc.) inside, and the PVCis affixed to the card using pre-formed tabs where the adhesive isplaced. The adhesive can be strong enough so that the pack may hang on apeg, but weak enough so that this way one can tear open the join andaccess the item. Sometimes with large items or multiple enclosed pills,tablets, geltabs, etc., the card has a perforated window for access. Inone aspect, more secure blister packs, e.g., for items such as pills,tablets, geltabs, etc. of the invention are used, and they can compriseof two vacuum-formed PVC sheets meshed together at the edges, with theinformative card inside.

In one aspect, blister packaging comprises at least two components(e.g., is a multi-ingredient combination of drugs of the invention): athermoformed “blister” which houses the product (e.g., a pharmaceuticalcombination of the invention), and then a “blister card” that is aprinted card with an adhesive coating on the front surface. During theassembly process, the blister component, which is most commonly made outof PVC, is attached to the blister card using a blister machine. Thismachine introduces heat to the flange area of the blister whichactivates the glue on the card in that specific area and ultimatelysecures the PVG blister to the printed blister card. The thermoformedPVG blister and the printed blister card can be as small or large.Conventional blister packs can also be sealed (e.g., using an AERGO 8DUO®, SCA Consumer Packaging, Inc., DeKalb, Ill.) using regular heatseal tooling. This alternative aspect, using heat seal tooling, can sealcommon types of thermoformed packaging.

As discussed herein, the products of manufacture of the invention cancomprise the packaging of the therapeutic drug combinations of theinvention, alone or in combination, as “blister packages” or as aplurality of packettes, including as lidded blister packages, liddedblister or blister card or packets, or a shrink wrap.

In one aspect, laminated aluminum foil blister packs are used, e.g., forthe preparation of drugs designed to dissolve immediately in the mouthof a patient. This exemplary process comprises having the drugcombinations of the invention prepared as an aqueous solution(s) whichare dispensed (e.g., by measured dose) into an aluminum (e.g., alufoil)laminated tray portion of a blister pack. This tray is then freeze-driedto form tablets which take the shape of the blister pockets. The alufoillaminate of both the tray and lid fully protects any highly hygroscopicand/or sensitive individual doses. In one aspect, the pack incorporatesa child-proof peel open security laminate. In one aspect, the systemgive tablets an identification mark by embossing a design into thealufoil pocket that is taken up by the tablets when they change fromaqueous to solid state. In one aspect, individual ‘push-through’ blisterpacks/packettes are used, e.g., using hard temper aluminum (e.g.,alufoil) lidding material. In one aspect, hermetically-sealed highbarrier aluminum (e.g., alufoil) laminates are used. In one aspect, anyof the invention's products of manufacture, including kits or blisterpacks, use foil laminations and strip packs, stick packs, sachets andpouches, peelable and non-peelable laminations combining foil, paper,and film for high barrier packaging.

Other means for containing said unit dosages can include bottles andvials, wherein the bottle or vial comprises a memory aid, such as aprinted label for administering said unit dosage or dosages. The labelcan also contain removable reminder stickers for placement on a calendaror dayminder to further help the patient to remember when to take adosage or when a dosage has been taken.

ORG 34517 Promotes Sensitivity to Chemotherapy

The glucocorticoid receptor (GR) is expressed in a subset of bothERalpha-positive and -negative human breast cancers as well as in someovarian cancers. In vitro and in vivo experiments suggest thatactivation of the GR in ER-negative pre-malignant breast epithelial andcancer cells initiates cell survival pathways under stress conditionsthat normally induce significant cell death (e.g. chemotherapy,radiation, growth factor deprivation). Thus, GR antagonism is predictedto enhance chemotherapy sensitivity of GR+/ER− breast cancer cells byblocking stress-mediated cell survival pathways that would otherwisecounteract chemotherapy-induced apoptosis.

In support of the present invention that GR activation mediateschemo-resistance (and associated increase risk of relapse for earlystage ER− breast cancers), recently the association was examined betweenGR (NR3C1) gene expression and GR target gene expression in human ER−breast cancers and cell lines. It was found that in ER− breast cancers,high GR expression is associated with a significantly increasedprobability of relapse in early stage patients. This analysis andprevious data in breast and ovarian cancer have led to the presentinvention wherein “GR high” breast tumors may respond better tocytotoxic therapies if glucocorticoid receptor (GR) antagonist therapyis administered with chemotherapy. Furthermore, GR antagonist therapyshould not worsen the lymphopenia sometimes associated with taxanetherapy because GR signaling is pro-apoptotic to lymphocytes.

Cancer/Tumor Stem Cells

Cancer stem cells comprise a unique subpopulation (often 0.1-10% or so)of a tumor that, relative to the remaining 90% or so of the tumor (i.e.,the tumor bulk), are more tumorigenic, relatively more slow-growing orquiescent, and often relatively more chemoresistant than the tumor bulk.Given that conventional therapies and regimens have, in large part, beendesigned to attack rapidly proliferating cells (i.e., those cancer cellsthat comprise the tumor bulk), cancer stem cells which are oftenslow-growing may be relatively more resistant than faster growing tumorbulk to conventional therapies and regimens. Cancer stem cells canexpress other features which make them relatively chemoresistant such asmulti-drug resistance and anti-apoptotic pathways. The aforementionedwould constitute a key reason for the failure of standard oncologytreatment regimens to ensure long-term benefit in most patients withadvanced stage cancers—i.e., the failure to adequately target anderadicate cancer stem cells. In some instances, a cancer stem cell(s) isthe founder cell of a tumor (i.e., it is the progenitor of the cancercells that comprise the tumor bulk).

Cancer stem cells have been identified in a large variety of cancertypes. For instance, Bonnet et al., using flow cytometry were able toisolate the leukemia cells bearing the specific phenotype CD34+CD38−,and subsequently demonstrate that it is these cells (comprising <1% of agiven leukemia), unlike the remaining 99+% of the leukemia bulk, thatare able to recapitulate the leukemia from which it was derived whentransferred into immunodeficient mice. See, e.g., Nat Med 3:730-737(1997). That is, these cancer stem cells were found as <1 in 10,000leukemia cells yet this low frequency population was able to initiateand serially transfer a human leukemia into severe combinedimmunodeficiency/non-obese diabetic (NOD/SCID) mice with the samehistologic phenotype as in the original tumor.

Cox et al. identified small subfractions of human acute lymphoblasticleukemia (ALL) cells which had the phenotypes CD34⁺/CD10⁻ andCD34⁺/CD19⁻, and were capable of engrafting ALL tumors inimmunocompromised mice—i.e. the cancer stem cells. In contrast, noengraftment of the mice was observed using the ALL bulk, despite, insome cases, injecting 10-fold more cells. See Cox et al., Blood 104(19):2919-2925 (2004).

Multiple myeloma was found to contain small subpopulations of cells thatwere CD138− and, relative to the large bulk population of CD138+ myelomacells, had greater clonogenic and tumorigenic potential. See Matsui etal., “Characterization of clonogenic multiple myeloma cells,” Blood103(6): 2332. The authors concluded that the CD138− subpopulation ofmultiple myeloma was the cancer stem cell population.

Kondo et al. isolated a small population of cells from a C6-glioma cellline, which was identified as the cancer stem cell population by virtueof its ability to self-renew and recapitulate gliomas inimmunocompromised mice. See Kondo et al., Proc. Natl. Acad. Sci. USA101:781-786 (2004). In this study, Kondo et al. determined that cancercell lines contain a population of cancer stem cells that confer theability of the line to engraft immunodeficient mice.

Breast cancers were shown to contain a small population of cells withstem cell characteristics (bearing surface markers CD44+CD24low lin−).See Al-Hajj et al., Proc. Natl. Acad. Sci. USA 100:3983-3988 (2003). Asfew as 200 of these cells, corresponding to 1-10% of the total tumorcell population, are able to form tumors in NOD/SCID mice. In contrast,implantation of 20,000 cells that lacked this phenotype (i.e. the tumorbulk) was unable to re-grow the tumor.

A subpopulation of cells derived from human prostate tumors was found toself-renew and to recapitulate the phenotype of the prostate tumor fromwhich they were derived thereby constituting the prostate cancer stemcell population. See Collins et al., “Prospective Identification ofTumorigenic Prostate Cancer Stem Cells,” Cancer Res 65(23):10946-10951(2005).

Fang et al. isolated a subpopulation of cells from melanoma with cancerstem cell properties. In particular, this subpopulation of cells coulddifferentiate and self-renew. In culture, the subpopulation formedspheres whereas the more differentiated cell fraction from the lesionswere more adherent. Moreover, the subpopulation containing sphere-likecells were more tumorigenic than the adherent cells when grafted intomice. See Fang et al., “A Tumorigenic Subpopulation with Stem CellProperties in Melanomas,” Cancer Res 65(20): 9328-9337 (2005).

Singh et al. identified brain tumor stem cells. When isolated andtransplanted into nude mice, the CD133+ cancer stem cells, unlike theCD133− tumor bulk cells, form tumors that can then be seriallytransplanted. See Singh et al., “Identification of human brain tumorinitiating cells,” Nature 432:396-401 (2004); Singh et al., “Cancer stemcells in nervous system tumors,” Oncogene 23:7267-7273 (2004); Singh etal., “Identification of a cancer stem cell in human brain tumors,”Cancer Res. 63:5821-5828 (2003).

Since conventional cancer therapies target rapidly proliferating cells(i.e., cells that form the tumor bulk) these treatments are believed tobe relatively ineffective at targeting and impairing cancer stem cells.In fact, cancer stem cells, including leukemia stem cells, have indeedbeen shown to be relatively resistant to conventional chemotherapeutictherapies (e.g. Ara-C, daunorubicin) as well as other targeted therapies(e.g. Gleevec®, Velcade®).

Cancer Therapies

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, is currently being used, or may be used for theprevention, treatment and/or management of cancer can be used toprevent, treat, and/or manage the patient whose neoplasia and/or cancerstem cells are monitored in accordance with the methods of theinvention. Also, such neoplasia and/or cancer stem cell monitoring canbe employed in conjunction with any therapy for cancer according to theinstant invention. Therapies (e.g., therapeutic or prophylactic agents)include, but are not limited to, peptides, polypeptides, fusionproteins, nucleic acid molecules, small molecules, mimetic agents,synthetic drugs, inorganic molecules, and organic molecules.Non-limiting examples of cancer therapies include chemotherapies,radiation therapies, hormonal therapies, anti-angiogenesis therapies,targeted therapies, and/or biological therapies includingimmunotherapies and surgery. In certain embodiments, a prophylacticallyand/or therapeutically effective regimen comprises the administration ofa combination of therapies. In certain embodiments, ORG 34517 can beadministered as an agent to treat or prevent neoplasia. In certainembodiments, RU486 (mifepristone) can be administered as an agent totreat or prevent neoplasia.

Examples of cancer therapies include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents,dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; histone deacetylase inhibitors(HDAC-Is) hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;imatinib mesylate (Gleevec, Glivec); interleukin II (includingrecombinant interleukin II, or rIL2), interferon alpha-2a; interferonalpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; lenalidomide (Revlimid); letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantronehydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride;anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; InternationalPublication No. WO 02/098370, which is incorporated herein by referencein its entirety)); megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mifepristone; mitindomide; mitocarcin; mitocromin;mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantronehydrochloride; mycophenolic acid; nocodazole; nogalamycin; ORG 34517;ormaplatin; oxaliplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; RU486;safingol; safingol hydrochloride; semustine; simtrazene; sparfosatesodium; sparsomycin; spirogermanium hydrochloride; spiromustine;spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; InternationalPublication No. WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; porfimer sodium;porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene;raf antagonists; raltitrexed; ramosetron; ras farnesyl proteintransferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptinedemethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RIIretinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginoneB1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim;Sdi 1 mimetics; semustine; senescence derived inhibitor 1; senseoligonucleotides; signal transduction inhibitors; signal transductionmodulators; gamma secretase inhibitors, single chain antigen bindingprotein; sizofuran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; 5-fluorouracil; leucovorin;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;thalidomide; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; anti-integrin antibodies (e.g., anti-integrin a_(v)b₃antibodies); vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

A non-limiting list of compounds that could be used to target cancerstem cells includes: inhibitors of interleukin-3 receptor (IL-3R) andCD123 (including peptides, peptide-conjugates, antibodies,antibody-conjugates, antibody fragments, and antibodyfragment-conjugates that target IL-3R or CD123); cantharidin;norcantharidin and analogs and derivatives thereof; Notch pathwayinhibitors including gamma secretase inhibitors; sonichedgehog/smoothened pathway inhibitors including cyclopamine and analogsthereof; antibodies to CD96; certain NF-kB/proteasome inhibitorsincluding parthenolide and analogs thereof; certain triterpenesincluding celastrol; certain mTOR inhibitors; compounds and antibodiesthat target the urokinase receptor; sinefungin; certain inosinemonophosphate dehydrogenase (IMPDH) inhibitors; PPAR-alpha andPPAR-gamma agonists and antagonists (including pioglitazone,tesaslitazar, muraglitazar, peliglitazar, lobeglitazone, balaglitazone,ragaglitazar, rosiglitazone, farglitazar, sodelglitazar, reglitazar,naveglitazar, oxeglitazar, metaglidasen, netoglitazone, darglitazone,englitazone, thiazolidinediones, aleglitazar, edaglitazone,rivoglitazone, troglitazone, imiglitazar, and sipoglitazar); telomeraseinhibitors; antibodies to EpCAM (ESA); GSK-3 beta agonists andantagonists (including Lithium, 6-bromoinirubin-3′-oxime (BIO), TDZD8);Wnt pathway inhibitors including antibodies to frizzled or smallmolecules that inhibit disheveled/frizzled or beta catenin; anti-CD20antibodies and conjugates (e.g. Rituxan, Bexxar, Zevalin) for novel usein multiple myeloma or melanoma; anti-CD133 antibody; anti-CD44antibody; antibodies to IL-4; certain differentiation agents such asversnarinone; compounds that target CD33 such as an antibody orbetulinic acid; compounds that target lactadherin such as an antibody;small molecules or antibodies that target CXCR4 or SDF-1; smallmolecules or antibodies that target multi-drug resistance pumps;inhibitors of survivin; inhibitors of XIAP; small molecules that targetBcl-2; antibodies to CLL-1; and furin inhibitors (such ascucurbitacins).

An additional non-limiting list of compounds that could also be used totarget cancer and/or cancer stem cells includes: i) antibodies, antibodyfragments, and proteins that are either naked or conjugated to atherapeutic moiety that target certain cell surface targets on cancerstem cells, or ii) small molecules known in the art including ones thatcan be further optimized (e.g., via chemistry) or identified via acancer stem cell-based screen (e.g., such as one that would determinewhether a compound impairs proliferation or viability of a cancer stemcell through standard methods, the cell surface and intracellulartargets including (not meant to be exhaustive) are: Rex1 (Zfp42), CTGF,Activin A, Wnt, FGF-2, HIF-1, AP-2gamma, Bmi-1, nucleostemin, hiwi,Moz-TIF2, Nanog, beta-arrestin-2, Oct-4, Sox2, stella, GDF3, RUNX3,EBAF, TDGF-1, nodal, ZFPY, PTNE, Evi-1, Pax3, Mc1-1, c-kit, Lex-1, Zfx,lactadherin, aldehyde dehydrogenase, BCRP, telomerase, CD133, Bcl-2,CD26, Gremlin, and FoxC2.

In some embodiments, the therapy(ies) is an immunomodulatory agent.Non-limiting examples of immunomodulatory agents include proteinaceousagents such as cytokines, peptide mimetics, and antibodies (e.g., human,humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2fragments or epitope binding fragments), nucleic acid molecules (e.g.,antisense nucleic acid molecules and triple helices), small molecules,organic compounds, and inorganic compounds. In particular,immunomodulatory agents include, but are not limited to, methotrexate,leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A,minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)),methylprednisolone (MP), corticosteroids, steroids, mycophenolatemofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamides (e.g., leflunamide), T cell receptor modulators,cytokine receptor modulators, and modulators mast cell modulators. Otherexamples of immunomodulatory agents can be found, e.g., in U.S.Publication No. 2005/0002934 A1 at paragraphs 259-275 which isincorporated herein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent.

In some embodiments, the therapy(ies) is an anti-angiogenic agent.Non-limiting examples of anti-angiogenic agents include proteins,polypeptides, peptides, fusion proteins, antibodies (e.g., human,humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments,F(ab)₂ fragments, and antigen-binding fragments thereof) such asantibodies that specifically bind to TNF-alpha, nucleic acid molecules(e.g., antisense molecules or triple helices), organic molecules,inorganic molecules, and small molecules that reduce or inhibitangiogenesis. Other examples of anti-angiogenic agents can be found,e.g., in U.S. Publication No. 2005/0002934 A1 at paragraphs 277-282,which is incorporated by reference in its entirety. In otherembodiments, the therapy(ies) is not an anti-angiogenic agent.

In certain embodiments, the therapy(ies) is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, chlorambucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, mephalen,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclins include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etopiside (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine). In someembodiments of the invention, the therapy(ies) includes theadministration cantharidin or an analog thereof. The invention includesthe use of agents that target cancer stem cells. In certain embodiments,the agent acts alone. In other embodiments, the agent is attacheddirectly or indirectly to another therapeutic moiety. Non-limitingexamples of therapeutic moieties include, but are not limited toalkylating agents, anti-metabolites, plant alkaloids, cytotoxic agents,chemotherapeutic agents (e.g., a steroid, cytosine arabinoside,fluoruracil, methotrexate, aminopterin, mitomycin C, demecolcine,etoposide, mithramycin, calicheamicin, CC-1065, chlorambucil ormelphalan), radionuclides, therapeutic enzymes, cytokines, toxinsincluding plant-derived toxins, fungus-derived toxins, bacteria-derivedtoxin (e.g., deglycosylated ricin A chain, a ribosome inactivatingprotein, alpha-sarcin, aspergillin, restirictocin, a ribonuclease, adiphtheria toxin, Pseudomonas exotoxin, a bacterial endotoxin or thelipid A moiety of a bacterial endotoxin), growth modulators and RNase.In some embodiments, the agent used is an agent that binds to a marker,e.g., an antigen on a cancer stem cell. In a specific embodiment, theagent binds to an antigen that is expressed at a greater level on cancerstem cells than on normal stem cells. In a specific embodiment, theagent binds specifically to a cancer stem cell antigen that is not anormal stem cell. In other embodiments, the therapy(ies) is an agentthat binds to a marker on cancer stem cells. In one embodiment, theagent that binds to a marker on cancer stem cells is an antibody or anantibody conjugated to a therapeutic moiety or an antibody fragmentconjugated to a therapeutic moiety.

For example, in a specific embodiment, the agent binds specifically tothe IL-3 Receptor (IL-3R). In some embodiments, the agent that binds tothe IL-3R is an antibody or an antibody fragment that is specific forIL-3R. In some embodiments, the antibody or antibody fragment isconjugated either chemically or via recombinant technology to atherapeutic moiety (e.g., a chemotherapeutic agent, a plant-, fungus- orbacteria-derived toxin, a radionuclide) using a linking agent to effecta cell killing response. In certain embodiments, the antibody,antibody-conjugate, antibody fragment, or antibody fragment-conjugatebinds to the .alpha.-subunit of IL-3R (i.e., the CD123 antigen). Inother embodiments, the antibody, antibody-conjugate, antibody fragment,or antibody fragment-conjugate binds to the IL-3R, containing both the.alpha. and .beta. subunits. Methods for preparing antibodies to IL-3Rand mimetics of antibodies to IL-3R are described in U.S. Pat. No.6,733,743 B2, which is incorporated herein by reference in its entirety.

In other embodiments, the agent that binds to a marker on cancer stemcells is a ligand. In some embodiments, the ligand is a cytokine thatbinds to a cytokine receptor on cancer stem cells. In a particularembodiment, the ligand is interleukin-3 (IL-3) which can be conjugatedto a therapeutic moiety that includes a chemotherapeutic agent, aplant-, fungus-, or bacteria-derived toxin, or a radionuclide. TheIL-3-conjugate prophylactic and/or therapeutic therapy or regimen can bein the form of a recombinant fusion protein in embodiments where theconjugate is a toxin and the toxin is a protein, such as diphtheriatoxin. Methods for preparing and isolating an IL-3-diphtheria toxinfusion protein (IL3DT) are described in Frankel et al., “Diphtheriatoxin fused to human interleukin-3 is toxic to blasts from patients withmyeloid leukemias,” Leukemia 14:576 (2000) and Urieto et al., ProteinExpression and Purification 33: 123-133 (2004), the disclosures of whichare incorporated by reference in their entireties.

In certain embodiments, antibodies or fragments thereof that bind to amarker on cancer stem cells are substantially non-immunogenic in thetreated subject. Methods for obtaining non-immunogenic antibodiesinclude, but are not limited to, chimerizing the antibody, humanizingthe antibody, and isolating antibodies from the same species as thesubject receiving the therapy. Antibodies or fragments thereof that bindto markers in cancer stem cells can be produced using techniques knownin the art. See, for example, paragraphs 539-573 of U.S. Publication No.2005/0002934 A1, which is incorporated by reference in its entirety.

In some embodiments, the therapy comprises the use of X-rays, gamma raysand other sources of radiation to destroy cancer stem cells and/orcancer cells. In specific embodiments, the radiation therapy isadministered as external beam radiation or teletherapy, wherein theradiation is directed from a remote source. In other embodiments, theradiation therapy is administered as internal therapy or brachytherapywherein a radioactive source is placed inside the body close to cancerstem cells, cancer cells and/or a tumor mass.

In some embodiments, the therapy used is a proliferation based therapy.Non-limiting examples of such therapies include a chemotherapy andradiation therapy as described supra.

Currently available therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006).

In a specific embodiment, cycling therapy involves the administration ofa first cancer therapeutic for a period of time, followed by theadministration of a second cancer therapeutic for a period of time,optionally, followed by the administration of a third cancer therapeuticfor a period of time and so forth, and repeating this sequentialadministration, i.e., the cycle in order to reduce the development ofresistance to one of the cancer therapeutics, to avoid or reduce theside effects of one of the cancer therapeutics, and/or to improve theefficacy of the cancer therapeutics.

When two prophylactically and/or therapeutically effective regimens areadministered to a subject concurrently, the term “concurrently” is notlimited to the administration of the cancer therapeutics at exactly thesame time, but rather, it is meant that they are administered to asubject in a sequence and within a time interval such that they can acttogether (e.g., synergistically to provide an increased benefit than ifthey were administered otherwise). For example, the cancer therapeuticsmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect, preferably in a synergistic fashion. Thecombination cancer therapeutics can be administered separately, in anyappropriate form and by any suitable route. When the components of thecombination cancer therapeutics are not administered in the samepharmaceutical composition, it is understood that they can beadministered in any order to a subject in need thereof. For example, afirst prophylactically and/or therapeutically effective regimen can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of the second cancer therapeutic, toa subject in need thereof. In various embodiments, the cancertherapeutics are administered 1 minute apart, 10 minutes apart, 30minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hoursapart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hoursto 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart,10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24hours apart or no more than 48 hours apart. In one embodiment, thecancer therapeutics are administered within the same office visit. Inanother embodiment, the combination cancer therapeutics are administeredat 1 minute to 24 hours apart.

In a specific embodiment, the combination therapies have the samemechanism of action. In another specific embodiment, the combinationtherapies each have a different mechanism of action.

Diagnostic System

The following are sample embodiments of the chemistry processconfiguration. They each consist of a plastic cartridge or glassreaction vessel with reagent dispensed inside. The user will remove theseal from the cap on the reaction vessel, insert the high void volumeswab with saliva sample into cartridge or glass reaction vessel andplace the assay solution into the portable fluorescent polarizationapparatus for mixing with reagent one and two for rapid response todetermine the level of the constituent e.g., salivary cortisol.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

This study is performed to obtain evidence for enhancement ofchemotherapy sensitivity of GR+/ER−/PR−/Her2-neu- breast cancer cells byblocking stress-mediated cell survival pathways with a GR antagonist. Inthe present pilot study, the GR antagonist ORG 34517, is investigated,in one dose, in combination with the chemotherapeutic agent Taxol. Boththe Taxol and ORG 34517 doses are selected based on previousexperiments.

Materials and Methods—

1×107 TNBC (Triple Negative Breast Cancer; MDA-MB-231) cells in 50 ul ofPBS (phosphate buffered saline) are injected into the mammary fat pad of4-5 week old CB-17 SCID mice (Taconic Labs). The mice are allowed toform xenografted breast tumors of approximately 200 mm3 volume. Whentumors reach an average of 200 mm3 (Week “0”), 15 mice are separatedinto the 3 treatment groups 1) vehicle, 2) paclitaxel (Taxol), 3) ORG34517+paclitaxel. The used paclitaxel dose is 10 mg/kg/day dissolved inEthanol/Sesame Oil; ORG 34517 is administered in the maximum solubledose of 20.5 mg/kg/day dissolved in Ethanol/Castor Oil. The mice receiveintraperitoneal injections (200 ul) of drugs for 5 consecutive days (day1-5). Tumors are measured three times per week. Tumor size is bothexpressed as absolute volume and as relative growth in comparison tobaseline (day 0).

Results—

Treatment of the animals is started when the tumor has a volume of atleast 100 mm3 The total experimental group consisted of 15 animals. In 6animals the tumor growth was considered not sufficient. These animalswere not treated which resulted in individual group sizes of N=3. In thecontrol situation the tumor grows within 13 days with about 800 mm3 (seeraw data). To correct for baseline differences, for each animal therelative tumor growth is calculated. Relative growth is defined as(tumor size day x−tumor size day 0)/tumor size day 0. Group averages forthe relative tumor growth are depicted in FIG. 1. In the controlcondition (Veh/Veh) the relative growth is 379%. This growth is reducedto 183% by taxol treatment (Veh/Taxo). A further reduction of the growthis induced by the addition of ORG 34517 (517/Taxo). The relative growthof this latter group is 100%.

A repeated measures ANOVA did reveal a significant time-effect(F5.36)=2.73; p=0.035), a significant treatment effect (F(2.36)=26.86;p=0.000) and a significant interaction (F(10.36)=2.36; p=0.029). Itshould be remembered that the data are not normally distributed andtherefore only for descriptive purposes one-sided Student-t tests wereperformed and revealed a significant difference (p<0.05) on day 4 and 11when comparing Veh/Veh and Veh/Taxo. Comparing Veh/Taxo and 517/Taxoshowed significances on day 4, 7, 9 and 13. This statistical analysisshould be considered explorative because of the pilot nature of thestudy.

The individual scores for the 517/Taxo treated animals are depicted inFIG. 2. All three animals show a decreased tumor growth in comparison toVeh/Taxo treatment (mean scores). In 2 out of 3 animals a tumorshrinkage is observed, during treatment.

In this pilot study, the glucocorticoid receptor antagonist ORG 34517(when administered intraperitoneally in a dose of 20.5 mg/kg/day for 5days) potentiated the chemotherapeutic effect of Taxol. While Taxolinjections reduced the tumor growth; Taxol+ORG 34517 induced a tumorshrinkage in 2 out of 3 animals, during treatment.

Example 2

Mice were implanted with cultured ER-GR+ human breast cancer cells. Astumor volume in each mouse approached the test threshold of 200 mm3, themice were randomized to receive intraperitoneal injections of vehiclealone, chemotherapy (Paclitaxel) alone, and chemotherapy and ORG 34517.Each group contained 3 mice. Results show significant differences inattained tumor volume (see FIG. 10).

Example 3

In conventional dosage forms, like a capsule or a tablet(11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-onesuffers from a low exposure, high dose variability, large food effectand non-dose linearity. Another dosage form than a conventional tabletor capsule may be able to circumvent these issues.

The solubility of(11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-oneis determined in different solvents. These solutions can be of use forclinical treatment with the advantages as mentioned above.

TABLE 1 Compound Solubility Acetone >10 mg/mL Acetonitrile >10 mg/mLArachis oil 2.46 mg/g Benzylalcohol 390 mg/g Castor oil 7.38 mg/g Castoroil:benzylbenzoate (90:10) 15.0 mg/g Castor oil:propylene glycol laurate(0.55:0.45; v:v) 9.4 mg/g Corn oil 2.58 mg/g Cremophor EL 23 mg/gDimethylsulfoxide >100 mg/mL Dog bile:phosphate buffer (pH = 6.5) (1:1)~0.08 mg/mL Ethanol >10 mg/mL Ethanol:PEG 400:Propylene glycol(10:50:40) 28.6 mg/mL Ethanol:PEG 400:Propylene glycol:water 14.0 mg/mL(10:40:40:10) Gelicure 44/14 100 mg/mL Glucofurol:water (50:50) 0.7mg/mL Glycoferol 118 mg/ml HPβCD, 10% in water 0.207 mg/mL HPβCD, 20% inwater 0.395 mg/mL HPβCD, 40% in water 0.8 mg/mL Medium Chainmonoglyceride/glycerol monocaprylate 28 mg/g Methanol >10 mg/mL Miglyol812 4.5 mg/g Mulgofen:water (5:95) 2 mg/mL Olive oil 2.26 mg/g PEG 150037 mg/mL PEG 400 34.4 mg/mL PEG 400:Water (60:40) 0.0006 mg/mLPolysorbate in water (0.5%) <LOD Propylene glycol 8.9 mg/mL Propyleneglycol 8.9 mg/mL Soy oil 2.70 mg/g Span 80 7 mg/g TPGS:PEG400:propyleneglycol (20:60:5) 0.9 mg/mL TPGS:PEG 400:propyleneglycol(12:17:55) 0.7 mg/mL TPGS:d-Alpha Tocopheryl Polyethylene Glycol 10001.1 mg/mL Succinate (20%, as delivered) Water 0.003 mg/mL Water - Sodiumlauryl sulphate (0.5% w/v) 0.240 mg/mL Water - Sodium lauryl sulphate(1.0% w/v) 0.377 mg/mL

Example 4

During toxicology studies also attempts were made to increase theexposure to Org 34517 (6). A rat study showed interesting results.Wistar rats 5 male and 5 female) were dosed orally with three differentformulations:

-   -   a gelatin/mannitol suspension (173.6 mg/kg)    -   a solution of Org 34517 in arachis oil/10% benzyl alcohol (38.4        mg/kg    -   a dispersion of Org 34517 in Gelucire 44/14 tablets (a lipid        based Self Emulsifying Drug Delivery System, 7.4 mg/tablet, 37        mg/kg).        Table 2 shows the dose normalized results for Cm and AUC for the        male and female rats. Although the data show a large difference        between male and female rats, both the Gelucire and the Arachis        oil show a much higher bioavailability than the gelatin mannitol        suspension. It must be said that the data of arachis oil showed        a much larger variation than the other two formulations (50%        versus 10%, not shown here).

TABLE 2 Dose normalized toxicokinetic data of 3 formulations of Org34517 in rats (M: Male, F: Female). Gelucire Arachis Oil/10% Gelatin44/14 benzylalcohol Mannitol C_(max) M [ng/ml · mg ORG 3.1 8.2 0.434517] C_(max) F [ng/ml · mg ORG 42.6 16.8 3.5 34517 AUC₀₋₂₄ M [ng ·hr/ml · mg 30.8 99.3 4.9 ORG 34517] AUC₀₋₂₄ F [ng · hr/ml · mg 427.4231.8 51.7 ORG 34517]

The formulations which show a better bioavailability than gelatinmannitol are thought to increase the solubility of Org 34517 in the GItract.

Table 3 shows a finely milled suspension (“nanosuspension”) Study inDOG, doses 5 mg/kg; 15 mg/kg; 45 mg/kg comparison of SD results.Relative bioavailability compares AUC₀₋₂₄. Two dogs were included in thetest. The nanosuspension increases the exposure of(11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one(see FIG. 11).

TABLE 3 Relative bioavailability in dogs: comparison of conventionalsuspension with finely milled suspension. AUC₀₋₂₄ AUC₀₋₂₄ (conventional(finely milled suspension) suspension) Dose ng · hr/ml ng · hr/mlF_(rel)  5 mg/kg 10105 13288 1.31 15 mg/kg 21687 23095 1.06 45 mg/kg118493 90536 0.76

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method for treating Cushing's syndrome in apatient characterized by hypercortisolism, comprising: selecting apatient in need of treatment for Cushing's syndrome characterized byhypercortisolism; administering to said patient a therapeuticallyeffective amount of a pharmaceutical composition comprising: at leastone glucocorticoid receptor (GCR) antagonist selected from the groupconsisting of ORG 34517, 11-(substituted phenyl)-estra-4, 9-dienederivatives, and 11-(substituted phenyl)-estra-4, 9-diene derivatives offormula 1

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which the heteroatom is connected to the phenyl group at theposition indicated with an asterisk, the ring being optionallysubstituted with one or more halogen atoms; R₁ is H or 1-oxo(1-4C)alkyl;R2 is H, (1-8C)alkyl, halogen or CF3; Xis selected from (H₂OH), O, andNOH; and the interrupted line represents an optional bond; and at leastone pharmaceutically acceptable excipient, thereby treating Cushing'ssyndrome characterized by hypercortisolism in the patient.
 2. The methodof claim 1, wherein the GCR antagonist is ORG
 34517. 3. The method ofclaim 1, wherein the pharmaceutical compound is formulated ormanufactured as a liquid, elixir, aerosol, spray, powder, tablet, pill,capsule, gel, geltab, nano-suspension, nanoparticle, extended releasedosage form, or topical formulation.
 4. The method of claim 1, themethod further comprising: a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring the parameter of the mixture todetermine a blank measurement; d) combining said test sample and buffermixture with a labeled ligand which binds cortisol, wherein the labeledligand is provided in a label unit, in the reaction unit to produce anassay solution or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)comparing the measured cortisol levels to a predetermined referencerange cortisol levels, wherein when the level of cortisol is elevatedrelative to the predetermined reference range, then the patient hasCushing's syndrome which involves elevated cortisol, which is suitablefor GCR antagonist therapy; h) administering a pharmaceuticalcomposition comprising a GCR antagonist.
 5. The method of claim 4,wherein the GCR antagonist is ORG
 34517. 6. The method of claim 4,wherein the test sample is obtained from the patient over consecutivedays.
 7. The method of claim 4, wherein the method is to determine thecircadian cycle of the cortisol levels in the patient, and thepredetermined time is selected from the group consisting of hourly,every 4 hours, every 6 hours, every 8 hours, and every 12 hours.
 8. Themethod of claim 4, wherein the predetermined reference range is amedically standard reference range.
 9. The method of claim 4, whereinthe predetermined reference range is the patient's previously measuredlevel.
 10. The method of claim 4, wherein the ligand is detectablylabeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, anaffinity tag, and an epitope tag.
 11. The method of claim 4, whereinsaid measuring of said parameter of said mixture and said assay solutionis performed using a method selected from spectroscopic, photochemical,radiochemical, biochemical, enzymatic, immunochemical, chemical labeldisplacement, surface plasmon resonance, fluorescence resonance energytransfer, fluorescence quenching, lateral flow, and fluorescencepolarization means.